Oligomeric compounds for the modulation of survivin expression

ABSTRACT

Oligonucleotides directed against the survivin gene are provided for modulating the expression of survivin. The compositions comprise oligonucleotides, particularly antisense oligonucleotides, targeted to nucleic acids encoding the survivin. Methods of using these compounds for modulation of survivin expression and for the treatment of diseases associated with either overexpression of survivin, expression of mutated survivin or both are provided. Examples of diseases are cancer such as lung, breast, colon, prostate, pancreas, lung, liver, thyroid, kidney, brain, testes, stomach, intestine, bowel, spinal cord, sinuses, bladder, urinary tract or ovaries cancers. The oligonucleotides may be composed of deoxyribonucleosides or a nucleic acid analogue such as for example locked nucleic acid or a combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/776,934 filed on Feb. 10, 2004, which claims the benefitunder 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No.60/446,372 filed on Feb. 10, 2003 and U.S. Provisional PatentApplication Ser. No. 60/523,591 filed on Nov. 19, 2003, the entireteachings of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of survivin. In particular, this invention relates tooligomeric compounds and preferred such compounds are oligonucleotides,which are specifically hybridisable with nucleic acids encodingsurvivin. The oligonucleotide compounds have been shown to modulate theexpression of survivin and pharmaceutical preparations thereof and theiruse as treatment of cancer diseases are disclosed.

BACKGROUND OF THE INVENTION

Cancer, a leading cause of death worldwide, comprises a group ofdiseases, which are caused by genetic disorders resulting from genomicinstability. It has been postulated that all cancer cells harbourdefects in several regulatory pathways, which govern normal cellproliferation and homeostasis. Theses defects result in acquirement ofvarious cancer cell specific hallmark capabilities (Hanahan andWeinberg, 2000, Cell 100, 57-70). One of these hallmarks of cancer isevasion of apoptosis or programmed cell death, an evolutionary conservedprogram of cellular suicide (Hengartner, 2000, Nature 407, 770-776.).Apoptosis is essential in fetal development by removal of cells notneeded any longer, and maintenance of homeostasis of adult tissues bybalancing cell production and cell elimination. Additionally, cellsexhibiting aberrant features like mutations or genomic damages inducedby infectious agents or drugs are removed in this way. In malignantcells this cellular surveillance is missing due to inhibition ofapoptosis, which results in extended cell viability increasing the riskof cellular transformation, accelerated disease progression andresistance to therapy (Evan and Vousden, 2001, Nature 411, 342-348.Therefore, manipulation of apoptosis has emerged as a new therapeuticstrategy for treatment of cancer (Nicholson D W, 2000, Nature 407,810-816).

Two signaling pathways leading to induction of apoptosis are known, theintrinsic or mitochondrial pathway, induced by environmental stress andchemotherapeutics, and the extrinsic or death receptor pathway, inducedby effector cells of the immune system (Ashkenazi and Dixit, 1998Science 281, 1305-1308; Green and Reed, 1998, Science 281, 1309-1312).Both pathways culminate with the activation of caspases, a family ofintracellular cystein proteases, which within minutes dismantle thecell's structures leading to rapid cell death (Cohen, 1997, Biochem J326, 1-16). Both, apoptosis promoting as well as inhibiting proteins areknown. The Bcl-2 protein family comprises both, pro- and anti-apoptoticproteins. Among the inhibitors of apoptosis, the evolutionary highlyconserved inhibitor of apoptosis protein (IAP) family comprises eightproteins in humans. One of them, survivin, has only recently beenidentified (Ambrosini et al., 1997, Nat. Med. 3, 917-921). Survivininhibits apoptosis downstream of Bcl-2 by directly or indirectlyinhibiting the effector caspase −3 and −7 intracellular proteasesresponsible or apoptosis (Shin et al., 2001, Biochemistry 40, 1117-1123)Recent evidence suggests that survivin directly controls the activationof the upstream acting caspase 9. A survivin Thr³⁴-Ala dominant negativemutant fails to induce apoptosis in mouse embryonic fibroblastsdeficient in apoptosome components Apaf-1 or caspase 9 (Blanc-Brude etal., 2003, Clin. Cancer Res. 9, 2683-2692) The hepatitis B X-interactingprotein (HBXIP) operates as a cofactor for phosphorylated survivinallowing it to bind and suppress activation of pro-caspase 9 (Marusawaet al., 2003, EMBO J. 22, 2729-2740). Other modes of action arediscussed, too (Beltrami et al., 2004, J. Biol. Chem. 279, 2077-2084).

Survivin has attracted great intention as novel therapeutic target,because it is selectively expressed in cancer cells and it is requiredfor their viability. Survivin is normally expressed duringembryogenesis. Apart from the thyme, CD34+ bone-marrow-derived stemcells, placenta and the basal colonic epithelium, survivin is notdetecTable in normal adult tissues, but is basically overexpressed inall transformed cells independent of their mitotic status. Expression isgenerally regulated in a cell-cycle dependent manner peaking at mitosis(Li et al. 1998, Nature 396, 580-584). Upregulation in G2/M phasecompared to interphase can be more than 40-fold. Also, increased proteinstability due to phosphorylation of Thr 34 by CDC2-cyclin-B1 may accountfor elevated survivin levels at mitosis. In the interphase, the proteinlevel declines due to ubiquitin dependent proteolysis (Zhao et al.,2000, J Cell Sci. 113, 4363-71) to basal levels. It has been suggestedthat overexpression of survivin in cancer cells counteracts a defaultinduction of apoptosis, overcomes the G2/M checkpoint and thus enforcesprogression of cells through mitosis (Li et al., 1998, Nature 396,580-584).

In cell culture systems, inhibition of cell death by overexpression ofsurvivin is well established (Ambrosini et al. 1997, Nat. Med. 3,917-921; Tamm et al. 1998, Cancer Res. 58, 5315-5320; Mahotka et al.,1999, Cancer Res. 59, 6097-6102).

In vivo, survivin's role as inhibitor of apoptosis has been demonstratedin transgenic mice expressing survivin in the skin, which inhibited UVBinduced apoptosis of the keratinocytes (Grossman at al., 2001, J. Clin.Invest. 108, 991-999). Apart from its role in cellular apoptosis,survivin plays a critical role in various aspects of mitosis. Forexample, knocking out survivin in homozygous survivin knock-out miceleads to 100% lethality (Uren et al. 2000, Curr. Biol. 10, 1319-1328;Conway et al., 2002, Gastroenterolgy 123, 619-631). Survivin has beenfound to be associated with various components of the mitotic apparatus,such as centrosomes, mictrotubules and the remnants of the spindleapparatus—the midbodies. Microtubule association is essential forsurvivin's anti-apoptotic action.

Survivin's dual role as apoptosis inhibitor and essential factor in celldivision was demonstrated by targeted downregulation of survivin bytransfecting HeLa cells with EPR-1 cDNA, which is complementary tosurvivin. Downregualtion of survivin by EPR-1 antisense resulted inincreased apoptosis and inhibition of cell proliferation (Ambrosini etal., 1998, J. Biol. Chem. 273, 11177-11182). Other hallmarks of survivinablation are mitotic arrest, polyploidy, defect centrosome replication,microtubule nucleation and mitotic spindle assembly/stability andinhibition of cell division. Most of these effects are exacerbated in ap53^(−/−) mutant background (Beltrami et al., 2004, J. Biol. Chem. 279,2077-2084; Carvalho et al, 2003, J. Cell. Sci. 116, 2987-2998; Lens etal., 2003, EMBO J. 22, 2934-2947). The pivotal role of survivin inmitosis is underscored by its association with the mitotic apparatus,including microtubules of the metaphase and anaphase spindle, andkinetochores of metaphase chromosomes (Beltrami et al., 2004, J. Biol.Chem. 279, 2077-2084). Survivin colocalizes with other chromosomalpassenger proteins such as INCENP and Aurora B (Carvalho et al, 2003, J.Cell. Sci. 116, 2987-2998; Lens et al., 2003, EMBO J. 22, 2934-2947).Kinase activity of Aurora B is dependent upon interaction with surviving(Chen et al., 2003, J. Biol. Chem. 278, 486-490). It has been suggestedthat Aurora B kinase activity is essential to cytokinesis providing amechanistic link between survivin and cell division (Chen et al., 2003,J. Biol. Chem. 278, 486-490). Two reports demonstrate that survivin isrequired for sustained the checkpoint arrest in response to lack oftension on kinetochores of sister chromatides. Survivin appears to beessential for the maintenance of checkpoint protein BubR1 at thekinetochores and mitotic arrest under protracted conditions of lack oftension at the kinetochores. Taxol, a microtubule stabilizing agent,allows attachment of microtubules to kinetochores, but preventsgeneration of tension. Normally taxol treated cells arrest in mitosisdue to checkpoint activation mediated by kinetochore associated BubR1.Taxol treated Survivin depleted cells however manage to complete mitosisafter a delay during which BubR1 is lost from the kinetochore. Nocodazolalso prevents tension forming at the kinetochores but by preventingmicrotubule attachment. However depletion of Survivin has no effect oncheckpoint activation and mitotic arrest induced by Nocodazol. Survivinis apparently not involved in monitoring kinetochore occupancy bymicrotubuli but instead required for checkpoint monitoring spindletension excerted on the kinetochore. (Carvalho et al, 2003, J. Cell.Sci. 116, 2987-2998; Lens et al., 2003, EMBO J. 22, 2934-2947). Moreoverit has been suggested that survivin serves as a crucial p53 dependentmitotic checkpoint protein required for genomic integrity andcytoprotection (Beltrami et al., 2004, J. Biol. Chem. 279, 2077-2084).Survivin may therefore be an important link between cell death and theregulation of cell division. Due to its dual role as inhibitor ofapoptosis and promoter of mitosis survivin is an important factor inonset and progression of cancer as well as resistance tochemotherapeutic agents.

Its clinical role in cancer has been emphasized by detection of highlevels of survivin in almost all tumour types. Elevated expression ofsurvivin in tumours is associated with poor prognosis, increased cancerrecurrence and resistance to therapy (Kawasaki et al., 1998, Cancer Res.58, 5071-5074; Adida et al., 1998, Lancet 351, 882-883). Interestingly,lung and breast tumours express the highest levels of survivin. Thesetumours are generally associated with unfavourable prognosis due toearly metastasizing and development of resistance to a number ofmechanistically unrelated chemotherapeutic agents. Downregulation ofsurvivin has been shown to sensitize tumor cells to DNA damaging agentssuch as etoposide (Li et al., 1999, Nature Cell Biology 1, 461-466; Olieet al., 2000, Cancer Res. 60, 2805-2809; Jiang et al., 2001, J. Cell.Biochem. 83, 342-354), cisplatin (Pennati et al., 2004, Oncogene 23,386-394), doxorubicin (Zhou et al., 2002, J. Pharmacol. Exp. Ther. 303,124-131) and radiotherapy (Pennati et al., 2003, J. Invest. Dermatol.120, 648-654; Asanuma et al., 2002, Jpn. J. Cancer Res. 93, 1057-1062).Survivin depleted cells are particularly senetive to texol is also truefor taxol (Zaffaroni et al., 2002, Cell. Mol. Life Sci. 59, 1406-1412;Ling et al., 2004, J. Biol. Chem. Epub ahead of print). Resistance totaxol and radiotherapy has been shown to correlate with the expressionlevel of survivin (Zaffaroni et al., 2002, Cell. Mol. Life Sci. 59,1406-1412; Rodel et al., 2003, Int. J. Radiat. Oncol. Biol. Phys. 55,1341-1347) and sublethal concentrations of taxol has been shown toupregulate survivin expression significantly in MCF-7 breast cancercells (Ling et al., 2004, J. Biol. Chem. Epub ahead of print). Survivinappears to be required for the function of the spindle checkpoint inresponse to taxol treatment (Carvalho et al, 2003, J. Cell. Sci. 116,2987-2998; Lens et al., 2003, EMBO J. 22, 2934-2947). In the absence ofsurvivin cells are therefore deprived of one of their natural resistancemechanisms that allows repair of the adverse effects of taxol onmitosis.

Interestingly, survivin also plays a critical role in angiogenesis.Survivin was found upregulated in angiogenically stimulated endotheliumin vitro and in vivo (O'Connor et al., 2000, Am. J. Pathol. 156,393-398; Tran et al., 1999, Biochem. Biophys. Res. Commun. 264,781-788). Antisense targeting of survivin caused endothelial apoptosisand rapid involution of capillary-like vessels in vitro (Mesri et al.,2001a, Am. J. Pathol. 158, 1757-1765). Injection into human breastcancer xenografts of an adenovirus expressing a dominant negativeversion of survivin inhibited growth of established tumors. This wasassociated with apoptosis of both tumor cells and endothelial cells anda significant reduction in tumor derived blood vessels (Blanc-Brude etal., 2003, Clin. Cancer Res. 9, 2683-2692). Chemotherapy andradiotherapy targets both tumor cells and the proliferating endothelialcells of the tumor vasculature. Vascular endothelial growth factor(VEGF) has been shown to significantly reduce the proapoptotic potencyof chemotherapy on vascular endothelial cells. This cytoprotection todrug toxicity has been linked to a VEGF mediated upregulation ofsurvivin expression. Suppression of survivin activity abrogates thecytoprotective effect of VEGF to drugs that interfere with microtubuledynamics (Taxol) and damage DNA as well as protection against tumornecrosis factor α (Tran et al., 2002, Proc. Natl. Acad. Sci. USA 99,4349-4354; Mesri et al., 2001a, Am. J. Pathol. 158, 1757-1765). Inaddition expression of a dominant negative survivin (T34A) protein inendothelial cells (HUVECC and DMVEC) resulted in massive induction ofapoptosis (Blanc-Brude et al., 2003, Clin. Cancer Res. 9, 2683-2692).

Targeting survivin is increasingly being mentioned as having a dualanticancer activity by inducing tumor cell apoptosis and suppression oftumor associated angiogenesis (Altieri D C, 2003, Oncogene 22,8581-8591).

Several therapeutic approaches using survivin as target have beeninitiated. The most promising ones comprise vaccination strategies, useof mutant survivin as dominant-negative antagonists, and application ofsurvivin specific antisense oligonucleotides. Application of areplication deficient adenovirus expressing a dominant negative survivinmutant protein (Thr34-Ala) caused inhibited tumour growth in threedistinct breast cancer xenograft models in mice. This adenovirus hasshown in vivo efficacy in a breast cancer xenograft models and inducedexpression of survivin (T34A) in melanoma cells inhibited tumor growthin a melanoma xenograft model (Blanc-Brude et al., 2003, Clin. CancerRes. 9, 2683-2692; Grossman et al., 2001 Proc. Natl. Sci. USA 98;635-640). In cell cultures apoptosis was increased by binding of mutantsurvivin to CDC2-cyclin-B1 and thus preventing phosphorylation ofwildtype survivin (Mesri et al., 2001b, J. Clin. Invest 108, 981-990).Some CDC2 antagonists like purvalanol A and flavopiridol, preventingsurvivin phospholylation, are currently being tested in clinical trialsin combination with taxol (Schwartz et al., 2002, J. Clin. Oncol. 20,2157-2170).

Several approaches using antisense oligonucleotides have shown thatanti-survivin antisense oligonucleotides downregulate survivin in cellcultures, induce apoptosis and sensitize lung cancer cells and HeLacells to the chemotherapeutic agent etoposide (Li et al., 1999, NatureCell Biology 1, 461-466; Olie et al., 2000, Cancer Res. 60, 2805-2809;Jiang et al., 2001, J. Cell. Biochem. 83, 342-354). Inhibition ofseveral cell lines with antisense oligo ISIS 28599, a mixed backbone2′-O-MOE wingmer, resulted in multinucleated cells and induction ofapoptosis (Chen at al., 2000, Neoplasia 2, 235-241). In a mouse liverregeneration model survivin mRNA was reduced 90% by the antisenseoligonucleotide ISIS 114926 (Proceedings of the American Association forCancer Research, vol. 42, 2001, abstract #2468). Intratumoral injectionof antisense oligonucleotide ISIS 23722 reduced had limited effect onthe growth rate of aggressive non-Hodgkin's lymphoma xengraft tumors inmice (Ansell et al., 2004, Leukemia—Epub ahead of print).

There are currently no therapeutic agents, which effectively inhibit thesynthesis of survivin. Therefore, there is a longfelt need for agentsinhibiting tumor cell growth by reducing survivin expression. InWO9822589 methods of modulating apoptosis with agents, that modulatesthe amount or activity of survivin and methods for reducing the severityof a pathological state mediated by survivin with such agents aredisclosed. Such an agent is a construct encoding the EPR-1 codingstrand, which is complementary to survivin but no specific antisenseoligos are disclosed. WO0164741 discloses a “tet-off” promoter systemregulating a survivin antisense mRNA transcript. However, thisapplication does not disclose any antisense oligonucleotides.

Most of the oligonucleotides currently in clinical trials are based onthe phosphorothioate chemistry from 1988, which was the first usefulantisense chemistry to be developed. However, as it has become clear inrecent years this chemistry has serious shortcomings that limit itsclinical use. These include low affinity for their target mRNA, whichnegatively affects potency and puts restrictions on how small activeoligonucleotides can be thus complicating manufacture and increasingtreatment costs. Also, their low affinity translate into pooraccessibility to the target mRNA thus complicating identification ofactive compounds. Finally, phosphorothioate oligonucleotides suffer froma range of side effects that narrow their therapeutic window.

To deal with these and other problems, much effort has been invested increating novel analogues with improved properties. As depicted in thescheme 1 below, these include wholly artificial analogues such as PNAand Morpholino and more conventional DNA analogues such asboranosphosphates, N3′-P5′ phosphoroamidates and several 2′ modifiedanalogues, such as 2′-F, 2′-O-Me, 2′-O-methoxyethyl (MOE) and2′-O-(3-aminopropyl) (AP). More recently hexitol nucleic acid (HNA),2′-F-arabino nucleic acid (2′-F-ANA) and D-cyclohexenyl nucleoside(CeNA) have been introduced.

Many of these analogues exhibit improved binding to complementarynucleic acids, improvements in bio-stability or they retain the abilityto recruit a cellular enzyme, RNAseH, which is involved in themode-of-action of many antisense compounds. None of them, however,combine all of these advantages and in many cases improvements in one ofthe properties compromise one or more of the other properties. Also, inmany cases new complications have been noted which seriously limits thecommercial value of some of the analogues. These include low solubility,complex oligomerisation chemistries, very low cellular up-take,incompatibility with other chemistries, etc.

Antisense oligonucleotides for modulation of survivin expression fortreatment of diseases are disclosed in WO0018781 and WO0157059. Theseoligonucleotides are all between 18-20 bp in length and designed withthe phosphorothioate or the MOE chemistry.

WO014655 discloses one single antisense oligonucleotide targetingSurvivin and it is a fully modified phosphorthioate with some MOEnucleosides. The MOE chemistry has several limitations. It has onlymodest affinity, which only manifests when several MOE's are inserted enblock into the oligo. MOE belongs to the family of 2′-modifications andit is well known, for this group of compound, that the antisenseactivity is directly correlated with RNA binding affinity in vitro. AMOE 20 bp gapmer (5MOE/PO-10PS-5MOE/PO) targeting c-raf has beenreported to have an IC₅₀ of about 20 nm in T24 cells and an MOE gapmertargeting PKC-a has been reported to have an IC₅₀ of 25 nm in A549cells. In comparison, phosphorthioate compounds used in antisenseexperiments typically exhibit IC₅₀ in the 150 nm range. (Stein, Kreig,Applied Antisense Oligonucleotide Technology, Wiley-Liss, 1988, p 87-90)

WO03027244, filed subsequent to the present invention, discloses a20-mer phosphorthioate antisense oligonucleotides targeting survivinwhich show down regulation at very high concentrations (for examplecompound 903 showed 51% protein reduction at 200 nM).

It is a principal object of the present invention to provide noveloligomeric compounds, against the survivin mRNA. The compounds of theinvention have been found to exhibit an decreased IC₅₀ (thus increasedactivity), thereby facilitating an effective treatment of a variety ofcancer diseases in which the expression of survivin is implied as acausative or related agent. As explained in the following, thisobjective is best achieved through the utilisation of a super highaffinity chemistry termed LNA (Locked Nucleic Acid).

The present invention is directed to oligomeric compounds, particularlyLNA antisense oligonucleotides, which are targeted to a nucleic acidencoding survivin and which modulate the expression of the survivin.This modulation was particularly a very potent down regulation survivinmRNA as well as elicitation of apoptotic response. The LNA-containingoligomeric compounds can be as low as an 8-mer and certainly highlyactive as a 16-mers, which is considerably shorter than the reportedantisense compounds targeting survivin. These 16-mer oligomericcompounds have an IC₅₀ in the sub-nanomolar range. The invention enablesa considerable shortening of the usual length of an antisense oligomers(from 20-25 mers to, e.g., 8-16 mers) without compromising the affinityrequired for pharmacological activity. As the intrinsic specificity ofan oligo is inversely correlated to its length, such a shortening willsignificantly increase the specificity of the antisense compound towardsits RNA target. Furthermore, it is anticipated that shorter oligomericcompounds have a higher biostability and cell permeability than longeroligomeric compounds. For at least these reasons, the present inventionis a considerable contribution to the art.

SUMMARY OF THE INVENTION

Survivin is essential to cell proliferation and involved in multiplephases of mitosis. It is involved in several checkpoints linking mitosiswith cell division and apoptosis. Survivin is a member of the inhibitorpf apoptosis (IAP) gene family that suppresses programmed cell death(apoptosis) (see FIG. 6). Increased survivin expression is observed inmost common human neoplasms, including colorectal cancer, bladdercancer, lung carcinoma, breast cancer, malignant gloma andhaematological cancers. Expression of survivin correlates with advancedgrade and invasiveness in several cancers. Survivin is undetectable orpresent at very low levels in normal differentiated tissues, makingsurvivin a preferred target in several human cancers.

A central aspect of the invention to provide a compound consisting offrom 8-50 nucleosides, wherein said compound comprises a subsequence ofat least 8 nucleosides, said subsequence being located within a sequenceselected those listed in Table 1 and 2.

One embodiment of the invention is, since the sequence of the humanegenome is available and the annotation of its genes rapidly progressing,to identify the shortest possible, unique sequences in the target mRNA.LNA containing oligomeric compounds of the invention have also beencompared to a number of 18-mers containing LNA and/or phosphorthioateswhich are iso-sequential to the antisense oligomer the ISIS 23722. Acomparison to the ISIS 23722 (being a 18-mer 4 MOE 10 phosphorthioatefollowed by 4 MOE) has also been performed.

Pharmaceutical and other compositions comprising the oligomericcompounds of the invention are also provided. Further provided aremethods of modulating the expression of survivin in cells or tissuescomprising contacting said cells or tissues with one or more of theoligomeric compounds or compositions of the invention. Also disclosedare methods of treating an animal or a human, suspected of having orbeing prone to a disease or condition, associated with expression ofsurvivin by administering a therapeutically or prophylacticallyeffective amount of one or more of the oligomeric compounds orcompositions of the invention. Further, methods of using oligomericcompounds for the inhibition of expression of survivin and for treatmentof diseases associated with survivin activity are provided. Examples ofsuch diseases are different types of cancer, such as for instance lung,breast, colon, prostate, pancreas, lung, liver, thyroid, kidney, brain,testes, stomach, intestine, bowel, spinal cord, sinuses, bladder,urinary tract or ovaries.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Illustration of the different designs of the invention: Gapmers,Head- and Tailmers and Mixmers of different composition. For the mixmer,the numbers designate the alternate contiguous stretch of DNA,β-D-oxy-LNA or α-L-LNA. In the drawing, the line is DNA, the gray shadowcorresponds to α-L-LNA residues and the rectangle is β-D-oxy-LNA.

FIG. 2 Survivin mRNA downregulation by LNA antisense oligomericcompound. Northern blot of total RNA from 15PC3 that have been treatedwith 0.2, 1, 5, 25 nM compound 2A, 6A, 9A, 15A respectively. Allcompounds were effective inhibitors at low concentrations.

FIG. 3 Survivin mRNA downregulation by LNA antisense oligomericcompound. Northern Blot of total RNA from SW480 (upper panel) and A549(lower panel) that have been treated with 0.2, 1, 5, 25 nM compound 2Aand 15A, respectively. Cells were transfected with oligonucleotide andcultured for 24 h.

FIG. 4 General scheme of the synthesis of thio-LNA.

FIG. 5 SEQ ID No 1 GenBank accession number NM_(—)001168 human survivinmRNA sequence.

FIG. 6 Schematic way of Survivin in the apoptotic pathway.

FIG. 7 Survivin mRNA downregulation by LNA antisense oligonucleotides.Cells were transfected with oligo nucleotide and cultures for 24 h.Total RNA was extracted and expression of Survivin mRNA were detected byeither Realtime PCR in 15PC3 and MCF-7. Survivin expression is presentednormalised to GAPDH transcript steady state.

FIG. 8 Induction of apoptosis by LNA containing antisense oligonucleotides. 15PC3 cells transfected with the oligos and concentrationsindicated in 96 well. 24 h following transfection Caspase 3/7-Gloreagens were added as described and the induction of luminescence(luciferase activity) were recorded in a Luminoskan Ascent instrumentfrom Thermo Labsystems. The luciferase activity is measured as RelativeLight Units per seconds (RLU/s).

FIG. 9. shows that the LNA containing compounds (145A and 145C) improvesinduction of apoptosis compared to the iso-sequential MOE compoundISIS27322 (here 145F) and the iso-sequential phosphorthioate compound(145D). Mismatch controls of a LNA compound (146C) and the MOE compound(146F) as well as the LNA compound 15B was also included in the study.The targeted downregulation of Survivin mRNA using LNA antisenseoligomeric compound results in increased apoptosis in 15PC3 cells.Activation of apoptosis is measured by cytometric bead array. Foldinduction is presented relative to mock treated cells.

FIG. 10 Using immunohistochemistry detection of active Caspase 3 wasdetected in 15PC3 cells treated with LNA antisense oligonucleotides 15Atargeting Survivin.

FIG. 11 LNA antisense inhibition of Survivin in proliferating cancercells. For example, compound 6A is particularly potent.

FIG. 12 Down regulation of Survivin in 15PC3 cells transfected withcompound 15A analyzed by western blotting at 100 nM.

DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding survivin. The modulation is ultimately achange in the amount of survivin produced. In one embodiment this isaccomplished by providing antisense compounds, which specificallyhybridise with nucleic acids encoding survivin. The modulation ispreferably an inhibition of the expression of survivin, which leads to adecrease in the number of functional proteins produced.

Antisense and other oligomeric compounds of the invention, whichmodulate expression of the target, are identified throughexperimentation or though rational design based on sequence informationon the target and know-how on how best to design an oligomeric compoundagainst a desired target. The sequences of these compounds are preferredembodiments of the invention. Likewise, the sequence motifs in thetarget to which these preferred oligomeric compounds are complementary(referred to as “hot spots”) are preferred sites for targeting.

The invention is directed to a compound consisting of 8-50 nucleotidesand/or nucleotide analogues, wherein said compound comprises asubsequence of at least 8 nucleotides or nucleotide analogues, saidsubsequence being located within a sequence selected from the groupconsisting of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143 and 144. The nucleotide analogues aretypically analogues of the nucleotides of the sequence SEQ ID NOS:2-144. Thus, the subsequence of the compound of the invention istypically located within a sequence selected from the group consistingof SEQ ID NOS: 2-144 or comprise analogues of the nucleotides within thesequence of SEQ ID NOS 2-144. A preferred nucleotide analogue of theinvention is LNA.

The total of 8-50 nucleotides and/or nucleotide analogues is intended tomean 8-50 nucleotides or 8-50 nucleotide analogues or a combinationthereof not exceeding a combined total of 50 nucleoside units.

In the present context, the term “nucleoside” is used in its normalmeaning, i.e. it contains a 2-deoxyribose unit or a ribose unit which isbonded through its number one carbon atom to one of the nitrogenousbases adenine (A), cytosine (C), thymine (T), uracil (U) or guanine (G).

In a similar way, the term “nucleotide” means a 2-deoxyribose unit orRNA unit which is bonded through its number one carbon atom to one ofthe nitrogenous bases adenine (A), cytosine (C), thymine (T) or guanine(G), uracil (U) and which is bonded through its number five carbon atomto an internucleoside phosphate group, or to a terminal group.

When used herein, the term “nucleotide analogue” refers to a non-naturaloccurring nucleotide wherein either the ribose unit is different from2-deoxyribose or RNA and/or the nitrogenous base is different from A, C,T and G and/or the internucleoside phosphate linkage group is different.Specific examples of nucleoside analogues are described by e.g. Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinionin Drug Development, 2000, 3(2), 293-213.

The terms “corresponding nucleoside analogue” and “correspondingnucleoside” are intended to indicate that the nucleobase in thenucleoside analogue and the nucleoside is identical. For example, whenthe 2-deoxyribose unit of the nucleotide is linked to an adenine, the“corresponding nucleoside analogue” contains a pentose unit (differentfrom 2-deoxyribose) linked to an adenine.

The term “nucleic acid” is defined as a molecule formed by covalentlinkage of two or more nucleotides. The terms “nucleic acid” and“polynucleotide” are used interchangeable herein

The term “nucleic acid analogue” refers to a non-natural nucleic acidbinding compound.

Nucleotide analogues and nucleic acid analogues are described in e.g.Freier & Altmann (Nucl. Acid Res., 1997, 25, 4429-4443) and Uhlmann(Curr. Opinion in Drug & Development (2000, 3(2): 293-213). Scheme 1illustrates selected examples of nucleotide analogues suitable formaking nucleic acids.

The term “LNA” refers to a nucleotide containing one bicyclic nucleosideanalogue, also referred to as a LNA monomer, or an oligonucleotidecontaining one or more bicyclic nucleoside analogues. LNA monomers aredescribed in WO 9914226 and subsequent applications, WO0056746,WO0056748, WO0066604, WO00125248, WO0228875, WO2002094250 andPCT/DK02/00488. One particular example of a thymidine LNA monomer is the(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-5-methyl-3-(thymin-1-yl)-2,5-dioxa-bicyclo[2:2:1]heptane.

The term “oligonucleotide” refers, in the context of the presentinvention, to an oligomer (also called oligo) or nucleic acid polymer(e.g. ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) or nucleicacid analogue of those known in the art, preferably Locked Nucleic Acid(LNA), or a mixture thereof. This term includes oligonucleotidescomposed of naturally occurring nucleobases, sugars and internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly or withspecific improved functions. A fully or partly modified or substitutedoligonucleotides are often preferred over native forms because ofseveral desirable properties of such oligonucleotides such as forinstance, the ability to penetrate a cell membrane, good resistance toextra- and intracellular nucleases, high affinity and specificity forthe nucleic acid target. The LNA analogue is particularly preferredexhibiting the above-mentioned properties.

By the term “unit” is understood a monomer.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 andso forth.

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in Scheme 2 is selected from S or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in Scheme 2 —N(H)—, N(R)—, CH₂—N(H)—, —CH₂—N(R)— where R isselected form hydrogen and C₁₋₄-alkyl. Amino-LNA can be in both beta-Dand alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in Scheme 2 represents —O— or —CH₂—O—. Oxy-LNA can be in bothbeta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in Scheme 2is —CH₂—O—.

By the term “alpha-L-LNA” comprises a locked nucleotide represented asshown in Scheme 3 (structure to the right).

By the term “LNA derivatives” comprises all locked nucleotide in Scheme2 except beta-D-methylene LNA e.g. thio-LNA, amino-LNA, alpha-L-oxy-LNAand ena-LNA.

The term “linkage group” is intended to mean a group capable ofcovalently coupling together two nucleosides, two nucleoside analogues,a nucleoside and a nucleoside analogue, etc. Specific and preferredexamples include phosphate groups and phosphorothioate groups.

In the present context the term “conjugate” is intended to indicate aheterogenous molecule formed by the covalent attachment of a compound asdescribed herein (i.e. a compound comprising a sequence of nucleosidesor nucleoside analogues) to one or more non-nucleotide ornon-polynucleotide moieties. Examples of non-nucleotide ornon-polynucleotide moieties include macromolecular agents such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically proteins may be antibodies for a targetprotein. Typical polymers may be polyethelene glycol.

The term “carcinoma” is intended to indicate a malignant tumor ofepithelial origin. Epithelial tissue covers or lines the body surfacesinside and outside the body. Examples of epithelial tissue are the skinand the mucosa and serosa that line the body cavities and internalorgans, such as intestines, urinary bladder, uterus, etc. Epithelialtissue may also extend into deeper tissue layers to from glands, such asmucus-secreting glands.

The term “sarcoma” is intended to indicate a malignant tumor growingfrom connective tissue, such as cartilage, fat, muscles, tendons andbones.

The term “glioma”, when used herein, is intended to cover a malignanttumor originating from glial cells

The term “a” as used about a nucleoside, a nucleoside analogue, a SEQ IDNO, etc. is intended to mean one or more. In particular, the expression“a component (such as a nucleoside, a nucleoside analogue, a SEQ ID NOor the like) selected from the group consisting of . . . ” is intendedto mean that one or more of the cited components may be selected. Thus,expressions like “a component selected from the group consisting of A, Band C” is intended to include all combinations of A, B and C, i.e. A, B,C, A+B, A+C, B+C and A+B+C.

In the present context, the term “C₁₋₄-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the chain hasfrom one to four carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

As used herein, the terms “target nucleic acid” encompass DNA encodingthe survivin, RNA (including pre-mRNA and mRNA) transcribed from suchDNA, and also cDNA derived from such RNA.

As used herein, the term “gene” means the gene including exons, introns,non-coding 5′ and 3′ regions and regulatory elements and all currentlyknown variants thereof and any further variants, which may beelucidated.

As used herein, the terms “oligomeric compound” refers to anoligonucleotide which can induce a desired therapeutic effect in humansthrough for example binding by hydrogen bonding to either a target gene“Chimeraplast” and “TFO”, to the RNA transcript(s) of the target gene“antisense inhibitors”, “siRNA”, “ribozymes” and oligozymes” or to theprotein(s) encoding by the target gene “aptamer”, spiegelmer” or“decoy”.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts, which maybe identified.

As used herein, the term “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe present invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

As used herein, the term “targeting” an antisense compound to aparticular target nucleic acid means providing the antisenseoligonucleotide to the cell, animal or human in such a way that theantisense compound are able to bind to and modulate the function of itsintended target.

As used herein, “hybridisation” means hydrogen bonding, which may beWatson-Crick, Holstein, reversed Holstein hydrogen bonding, etc. betweencomplementary nucleoside or nucleotide bases. Watson and Crick showedapproximately fifty years ago that deoxyribo nucleic acid (DNA) iscomposed of two strands which are held together in a helicalconfiguration by hydrogen bonds formed between opposing complementarynucleobases in the two strands. The four nucleobases, commonly found inDNA are guanine (G), adenine (A), thymine (T) and cytosine (C) of whichthe G nucleobase pairs with C, and the A nucleobase pairs with T. In RNAthe nucleobase thymine is replaced by the nucleobase uracil (U), whichsimilarly to the T nucleobase pairs with A. The chemical groups in thenucleobases that participate in standard duplex formation constitute theWatson-Crick face. Hoogsteen showed a couple of years later that thepurine nucleobases (G and A) in addition to their Watson-Crick face havea Hoogsteen face that can be recognised from the outside of a duplex,and used to bind pyrimidine oligonucleotides via hydrogen bonding,thereby forming a triple helix structure.

In the context of the present invention “complementary” refers to thecapacity for precise pairing between two nucleotides or nucleosidesequences with one another. For example, if a nucleotide at a certainposition of an oligonucleotide is capable of hydrogen bonding with anucleotide at the corresponding position of a DNA or RNA molecule, thenthe oligonucleotide and the DNA or RNA are considered to becomplementary to each other at that position. The DNA or RNA and theoligonucleotide are considered complementary to each other when asufficient number of nucleotides in the oligonucleotide can formhydrogen bonds with corresponding nucleotides in the target DNA or RNAto enable the formation of a sTable complex. To be stable in vitro or invivo the sequence of an antisense compound need not be 100%complementary to its target nucleic acid. The terms “complementary” and“specifically hybridisable” thus imply that the antisense compound bindssufficiently strongly and specifically to the target molecule to providethe desired interference with the normal function of the target whilstleaving the function of non-target mRNAs unaffected.

The oligomeric compounds according to the invention are potentmodulators of target. For example, in vitro inhibition of target isshown in Table 1 measured by Real time PCR. FIG. 2 shows in vitropotency of oligomeric compounds according to the invention measured byNorthern Blot. Very low IC₅₀ values of oligomeric compounds are shown inTable 3. All the above-mentioned experimental observations show that thecompounds according to the invention can constitute the active compoundin a pharmaceutical composition.

The subsequence of the compound of the invention is typically locatedwithin a sequence selected from the group consisting of SEQ ID NOS: 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 and 133. Theanalogues of the nucleotides are suitably of nucleotides within thethese sequences.

Typically, the compound of the invention comprises from 8-40nucleotides, more typically 8-35 nucleotides, even more typically 8-30nucleotides, suitably 8-25 nucleotides, more suitably 8-20 nucleotides,most suitably 12-20 nucleotides, such as 12, 13, 14, 15, 16, 17, 18, 19or 20 nucleotides. In a highly attractive embodiment of the invention,the compound of the invention comprises 14-18 nucleotides, such as 14,15, 16, 17 or 18 nucleotides, preferably 15-17 nucleotides, such as 15,16 or 17 nucleotides, more typically 15 nucleotides, or 16 nucleotides,or 17 nucleotides.

In a suitable embodiment of the invention, the subsequence within thesequence of SEQ ID NOS: 2-144 is typically at least 8 nucleotides ornucleotide analogues, such as at least 9 nucleotides from within thesequence or nucleotide analogues of the nucleotides within saidsequences. More typically, the subsequence is of at least 12 nucleotidesor nucleotide analogues from within said sequences, such as at least 14nucleotides or nucleotide analogues, such as 10, 11, 12, 13 14 15 or 16nucleotides or nucleotide analogues.

The nucleotides are typically linked to each other by means of a linkagegroup selected from the group consisting of a phosphate group, aphosphorothioate group and a boranophosphate group. Suitably, some orall of the nucleotides are linked to each other by means of a phosphategroup. Suitably, all nucleotides are linked to each other by means of aphosphate group.

Similarly, the nucleotides of the invention are typically linked to eachother by means of a linkage group selected from the group consisting ofa phosphate group, a phosphorothioate group and a boranophosphate group.

Preferred oligomeric compounds according to the invention are SEQ ID NO2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144 and their sequences are presented in Table 1.

In another embodiment of the invention, said nucleotides are linked toeach other by means of a phosphorothioate group, such as all nucleotidesbeing linked to each other by means of a phosphorothioate group. Aninteresting embodiment of the invention is directed to compounds of SEQNO 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, and 144 wherein each linkage group within each compound is aphosphorothioate group. Such modifications is denoted by the subscriptS. Alternatively stated, one aspect of the invention is directed tocompounds of SEQ NO 147, 151, 155, 159, 163, 167, 171, 175, 179, 183,187, 191, 195, 199, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240,244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296,300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352,356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408,412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464,468, 472, 476, 480, 484, 488, 492, 496, 500, 504, 508, 512, 516, 520,524, 528, 532, 536, 540, 544, 548, 552, 556, 560, 564, 568, 572, 576,580, 584, 588, 592, 596, 600, 604, 608, 612, 616, 620, 624, 628, 632,636, 640, 644, 648, 652, 656, 660, 664, 668, 672, 676, 680, 684, 688,692, 696, 700, 704, 708, 712 and 716.

A preferred subset of embodiments of the invention are compoundscomprising sequences of the formula SEQ ID NOS 147, 155, 163, 175, 199,612, 620, 632, 652, 656 and 664.

A further aspect of the invention is directed to compounds of SEQ NOS148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200,205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257,261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313,317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369,373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425,429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481,485, 489, 493, 497, 501, 505, 509, 513, 517, 521, 525, 529, 533, 537,541, 545, 549, 553, 557, 561, 565, 569, 573, 577, 581, 585, 589, 593,597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649,653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 697, 701, 705,709, 713 and 717.

A preferred subset of embodiments of the invention are compoundscomprising sequences of the SEQ ID NOS 613, 617, 621, 625, 629, 633,653, 657, 661 and 665.

A further aspect of the invention is directed to compounds of SEQ NOS149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201,206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258,262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310, 314,318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370,374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426,430, 434, 438, 442, 446, 450, 454, 458, 462, 466, 470, 474, 478, 482,486, 490, 494, 498, 502, 506, 510, 514, 518, 522, 526, 530, 534, 538,542, 546, 550, 554, 558, 562, 566, 570, 574, 578, 582, 586, 590, 594,598, 602, 606, 610, 614, 618, 622, 626, 630, 634, 638, 642, 646, 650,654, 658, 662, 666, 670, 674, 678, 682, 686, 690, 694, 698, 702, 706,710, 714 and 718.

A further aspect of the invention is directed to compounds of SEQ NOS150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202,207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259,263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315,319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371,375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427,431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483,487, 491, 495, 499, 503, 507, 511, 515, 519, 523, 527, 531, 535, 539,543, 547, 551, 555, 559, 563, 567, 571, 575, 579, 583, 587, 591, 595,599, 603, 607, 611, 615, 619, 623, 627, 631, 635, 639, 643, 647, 651,655, 659, 663, 667, 671, 675, 679, 683, 687, 691, 695, 699, 703, 707,711, 715 and 719.

A further aspect of the invention is directed to compounds of (SEQ IDNO: 203).

In an interesting embodiment, the compound of the invention comprisessequence 15E.

In a preferred embodiment, the compound of the invention comprises from8-50 nucleotides, wherein said compound comprises a subsequence of atleast 8 nucleotides, said subsequence being located within a sequenceselected from the group consisting of SEQ ID NOS: 2-144, wherein atleast one nucleotide is replaced by a corresponding nucleotide analogue.Typically, the compound of the invention comprises 1-50 nucleotideanalogues, such as 2-45 nucleotide analogues, 3-40 nucleotide analogues,suitably 4-35 nucleotide analogues, 5-30 nucleotide analogues, 6-25nucleotide analogues, typically 6-20 nucleotide analogues, moretypically 6-14 nucleotide analogues, such as 6-12 nucleotide analogues,such as 6, 7, 8, 9, 10, 11 or 12 nucleotide analogues.

The inventors have found that compounds of the invention comprising from6-16 nucleotide analogues with a different ribose unit suffice to haveimproved affinity over nucleotides. Thus, an interesting aspect of theinvention relates to a compound of the invention comprising 6-10, suchas 6, 7, 8, 9 or 10 nucleotide analogues with a different ribose unit,preferably 7, 8 or 9 nucleotide analogues with a different ribose unit,most typically 8 nucleotide analogues with a different ribose unit.Preferably, the nucleotide analogues with a different ribose unit is LNA

The present inventors have further found that nucleotide analogues witha different ribose units and furthermore with a modified internucleosidelinkade have a further improved effect for purposes of antisensemodifications. Thus, the 6-16 nucleotide analogues may have a modifiedribose unit, a different linkage group, or both.

Suitably, all nucleotides are replaced by a corresponding nucleotideanalogues.

A preferred nucleotide analogue of the invention is LNA.

A further preferred nucleotide analogue of the invention is wherein theinternucleoside phosphate linkage is a phosphorothioate.

A still further preferred nucleotide analogue is wherein the nucleotideis LNA with an internucleoside phosphorothioate linkage.

In an interesting embodiment, the compound of the invention comprisesfrom 8-50 nucleotides, wherein said compound comprises a subsequence ofat least 8 nucleotides, said subsequence being located within a sequenceselected from the group consisting of SEQ ID NOS: 2-144, wherein atleast one nucleotide is replaced by a corresponding nucleotide analogueand wherein the 3′ end comprises nucleotide, rather than a nucleotideanalogue.

In a particularly interesting embodiment, the compound comprises atleast one of nucleotide analogues, wherein said nucleotide analogue is alocked nucleic acid (LNA) of the formula

wherein Z and Z* are independently absent, selected among aninternucleoside linkage, a terminal group or a protecting group; whereinX and Y are independently selected from the group consisting of O, S,NR, CH₂, CH, (if part of a double bond), CH₂—O, CH₂—S, CH₂—NR, CH₂—CH₂,CH₂—CH (if part of a double bond) and CH═CH, where R is hydrogen orC₁₋₄-alkyl. The bonds represent connection to the linkage group.Typically, X is O and Y are independently selected from the groupconsisting of O, S and NR, where R is hydrogen or C₁₋₄-alkyl. Moretypically, X is O and Y is selected from the group consisting of O, Sand NH. Most typically, X is O and Y is O. In embodiments wherein atleast one of the LNA nucleotides is at the 3′-end, at said position Z isa terminal group and Z* is an internucleoside linkage. In embodimentswherein at least one of the LNA nucleotides is at the 5′-end, at saidposition Z is absent and Z* is a terminal group. Within the nucleotidesequence, Z is absent and Z* is an internucleoside linkage

In a suitable embodiment of the invention comprising LNA as thenucleotide analogues, said LNA is in the β-D or alpha-L also form,preferably in the β-D form.

In embodiments of the invention comprising at least one LNA as thenucleotide analogues, such as 1-50 LNA nucleotide analogues, such as2-45 LNA nucleotide analogues, 3-40 LNA nucleotide analogues, suitably4-35 LNA nucleotide analogues, 5-30 LNA nucleotide analogues, 6-25 LNAnucleotide analogues, typically 6-20 LNA nucleotide analogues, moretypically 6-14 LNA nucleotide analogues, such as 6-12 LNA nucleotideanalogues, such as 6, 7, 8, 9, 10, 11 or 12 LNA nucleotide analoguessaid nucleotides and/or nucleotide analogues are linked to each other bymeans of a linkage group selected from the group consisting of aphosphate group, a phosphorothioate group and a boranophosphate group.In a suitable embodiment of the invention comprising LNA nucleotideanalogues, said nucleotides and/or nucleotide analogues are linked toeach other by means of a phosphate group. In a preferred embodiment ofthe invention comprising LNA nucleotide analogues said nucleotidesand/or nucleotide analogues are linked to each other by means of aphosphorothioate group.

In a combination of interesting embodiment, in embodiments of theinvention comprising LNA nucleotide analogues said nucleotide and/ornucleotide analogues are linked to each other by means of aphosphorothioate group, wherein X is O and Y is O, and said LNA is inthe β-D form.

In embodiments of the compound of the invention comprising from 8-50nucleotides, wherein said compound comprises a subsequence of at least 8nucleotides, said subsequence being located within a sequence selectedfrom the group consisting of SEQ ID NOS: 2-144 and said nucleotidescomprising LNA nucleotide analogues, the subsequence typically maycomprise a stretch of 2-6 LNAs, as defined herein, followed by a stretchof 4-12 nucleotides, which is followed by a stretch of 2-6 LNAs, asdefined herein.

Subsequences comprising a stretch of LNAs, followed by a stretch ofnucleotides, followed by a stretch of LNAs are known as gapmers.

Suitably, said subsequence comprises a stretch of 4 LNAs, as definedherein, followed by a stretch of 8 nucleotides, which is followed by astretch of 4 LNAs as defined herein.

In embodiments of the compound of the invention comprising from 8-50nucleotides, wherein said compound comprises a subsequence of at least 8nucleotides, said subsequence being located within a sequence selectedfrom the group consisting of SEQ ID NOS: 2-144 and said 8-50 nucleotidescomprising LNA nucleotide analogues, said subsequence may comprises astretch of 2-6 LNAs as defined herein, followed by a stretch of 4-12nucleotides, which is followed by a stretch of 2-5 LNAs as definedherein, which is followed by 1-4 nucleotides, such as 1 or 2nucleotides, more typically a single nucleoside. The 1-4 nucleotides, 1or 2 nucleotides or single nucleotide is typically located at the 3′ endof the subsequence and more typically at the 3′ end of the oligomer.

In embodiments of the compound of the invention comprising from 8-50nucleotides, wherein said compound comprises a subsequence of at least 8nucleotides, said subsequence being located within a sequence selectedfrom the group consisting of SEQ ID NOS: 2-144 and said nucleotidescomprising LNA nucleotide analogues, said subsequence may typicallycomprise a stretch of 4 LNAs as defined herein, followed by a stretch of8 nucleotides, which is followed by a stretch of 3 LNAs as definedherein, which is followed by a single natural nucleotide. The singlenucleotide is typically located at the 3′ end of the subsequence andmore typically at the 3′ end of the oligomer.

In embodiments of the compound of the invention comprising from 8-50nucleotides, wherein said compound comprises a subsequence of at least 8nucleotides, said subsequence being located within a sequence selectedfrom the group consisting of SEQ ID NOS: 2-144 and said nucleotidescomprising LNA nucleotide analogues, said subsequence comprising astretch of LNAs, followed by a stretch of nucleotides, which is followedby a stretch of LNAs as defined herein as gapmers, said nucleotidesand/or LNAs are linked to each other by means of a linkage groupselected from the group consisting of a phosphate group, aphosphorothioate group and a boranophosphate group.

Suitably, said nucleotides and/or said LNAs are linked together by meansof phosphate groups. Typically, said nucleotides and/or said LNAs arelinked together by means of phosphorothioate groups.

In embodiments of the compound of the invention comprising a total offrom 8-50 nucleotides and/or nucleotide analogues, wherein said compoundcomprises a subsequence of at least 8 nucleotides, said subsequencebeing located within a sequence selected from the group consisting ofSEQ ID NOS: 2-144 and wherein said subsequence may consist of a stretchof 4 LNAs, as defined herein, a stretch of 8 nucleotides, and a stretchof 4 LNAs, as defined herein, so as to make a total of 16 nucleotidesand nucleotide analogues in said subsequence, said nucleotides and saidLNAs are linked together by means of phosphorothioate groups.

In a suitable embodiment, the subsequence is SEQ ID NO: 147. In asuitable embodiment, the subsequence is SEQ ID NO: 151. In a suitableembodiment, the subsequence is SEQ ID NO:155. In a suitable embodiment,the subsequence is SEQ ID NO: 159. In a suitable embodiment, thesubsequence is SEQ ID NO: 163. In a suitable embodiment, the subsequenceis SEQ ID NO: 167. In a suitable embodiment, the subsequence is SEQ IDNO: 171. In a suitable embodiment, the subsequence is SEQ ID NO: 175. Ina suitable embodiment, the subsequence is SEQ ID NO: 179. In a suitableembodiment, the subsequence is SEQ ID NO: 183. In a suitable embodiment,the subsequence is SEQ ID NO: 187. In a suitable embodiment, thesubsequence is SEQ ID NO: 191. In a suitable embodiment, the subsequenceis SEQ ID NO: 195. In a suitable embodiment, the subsequence is SEQ IDNO: 199. In a suitable embodiment, the subsequence is SEQ ID NO: 608. Ina suitable embodiment, the subsequence is SEQ ID NO: 612. In a suitableembodiment, the subsequence is SEQ ID NO: 616. In a suitable embodiment,the subsequence is SEQ ID NO: 620. In a suitable embodiment, thesubsequence is SEQ ID NO: 624. In a suitable embodiment, the subsequenceis SEQ ID NO: 628. In a suitable embodiment, the subsequence is SEQ IDNO: 632. In a suitable embodiment, the subsequence is SEQ ID NO: 636. Ina suitable embodiment, the subsequence is SEQ ID NO: 640. In a suitableembodiment, the subsequence is SEQ ID NO: 644. In a suitable embodiment,the subsequence is SEQ ID NO: 648. In a suitable embodiment, thesubsequence is SEQ ID NO: 652. In a suitable embodiment, the subsequenceis SEQ ID NO: 656. In a suitable embodiment, the subsequence is SEQ IDNO: 660. In a suitable embodiment, the subsequence is SEQ ID NO: 664. Ina suitable embodiment, the subsequence is SEQ ID NO: 668. In a suitableembodiment, the subsequence is SEQ ID NO: 672. In the immediatelyaforementioned individual suitable embodiments wherein the subsequenceis one selected from SEQ ID NOS: 148, 152, 156, 160, 164, 168, 172, 176,180, 184, 188, 192, 196, 200, 205, 209, 213, 217, 221, 225, 229, 233,237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289,293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345,349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401,405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457,461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513,517, 521, 525, 529, 533, 537, 541, 545, 549, 553, 557, 561, 565, 569,573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625,629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681,685, 689, 693, 697, 701, 705, 709, 713 and 717, the 3′ end LNA of thesubsequence may suitably be replaced by the corresponding nucleotide.

In a further suitable embodiment, the compound of the invention is asequence selected from the group consisting of SEQ ID NOS: 2-144 andwherein said sequence consisting of a stretch of 4 LNAs, as definedherein, a stretch of 8 nucleotides, and a stretch of 4 LNAs, as definedherein, so as to make a total of 16 nucleotides and nucleotide analoguesin said compound, said nucleotides and said LNAs being linked togetherby means of phosphorothioate groups.

In a suitable embodiment, the compound consists of SEQ ID NO:147. In asuitable embodiment, the compound consists of SEQ ID NO: 151. In asuitable embodiment, the compound consists of SEQ ID NO: 155. In asuitable embodiment, the compound consists of SEQ ID NO: 159. In asuitable embodiment, the compound consists of SEQ ID NO: 163. In asuitable embodiment, the compound consists of SEQ ID NO: 167. In asuitable embodiment, the compound consists of SEQ ID NO: 171. In asuitable embodiment, the compound consists of SEQ ID NO: 175. In asuitable embodiment, the compound consists of SEQ ID NO: 179. In asuitable embodiment, the compound consists of SEQ ID NO: 183. In asuitable embodiment, the compound consists of SEQ ID NO: 187. In asuitable embodiment, the compound consists of SEQ ID NO: 191. In asuitable embodiment, the compound consists of SEQ ID NO: 195. In asuitable embodiment, the compound consists of SEQ ID NO: 199. In asuitable embodiment, the compound consists of SEQ ID NO: 608. In asuitable embodiment, the compound consists of SEQ ID NO: 612. In asuitable embodiment, the compound consists of SEQ ID NO: 616. In asuitable embodiment, the compound consists of SEQ ID NO: 620. In asuitable embodiment, the compound consists of SEQ ID NO: 624. In asuitable embodiment, the compound consists of SEQ ID NO: 628. In asuitable embodiment, the compound consists of SEQ ID NO: 632. In asuitable embodiment, the compound consists of SEQ ID NO: 636. In asuitable embodiment, the compound consists of SEQ ID NO: 640. In asuitable embodiment, the compound consists of SEQ ID NO: 644. In asuitable embodiment, the compound consists of SEQ ID NO: 648. In asuitable embodiment, the compound consists of SEQ ID NO: 652. In asuitable embodiment, the compound consists of SEQ ID NO: 656. In asuitable embodiment, the compound consists of SEQ ID NO: 660. In asuitable embodiment, the compound consists of SEQ ID NO: 664. In asuitable embodiment, the compound consists of SEQ ID NO: 668. In asuitable embodiment, the compound consists of SEQ ID NO: 672. In theimmediately aforementioned individual suitable embodiments wherein thecompound is one selected from SEQ ID NOS: 147, 151, 155, 159, 163, 167,171, 175, 179, 183, 187, 191, 195, 199, 204, 208, 212, 216, 220, 224,228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280,284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336,340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392,396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448,452, 456, 460, 464, 468, 472, 476, 480, 484, 488, 492, 496, 500, 504,508, 512, 516, 520, 524, 528, 532, 536, 540, 544, 548, 552, 556, 560,564, 568, 572, 576, 580, 584, 588, 592, 596, 600, 604, 608, 612, 616,620, 624, 628, 632, 636, 640, 644, 648, 652, 656, 660, 664, 668, 672,676, 680, 684, 688, 692, 696, 700, 704, 708, 712 and 716, the 3′ end LNAof the compound may suitably be replaced by the correspondingnucleotide.

A further aspect of the invention relates to a conjugate comprising thecompound as defined herein at least one non-nucleotide ornon-polynucleotide moiety covalently attached to said compound.

In a related aspect of the invention, the compound of the invention islinked to ligands so as to form a conjugates said ligands intended toincrease the cellular uptake of the conjugate relative to the antisenseoligonucleotides. This conjugation can take place at the terminalpositions 5′/3′-OH but the ligands may also take place at the sugarsand/or the bases. In particular, the growth factor to which theantisense oligonucleotide may be conjugated, may comprise transferrin orfolate. Transferrin-polylysine-oligonucleotide complexes orfolate-polylysine-oligonucleotide complexes may be prepared for uptakeby cells expressing high levels of transferrin or folate receptor. Otherexamples of conjugates/lingands are cholesterol moieties, duplexintercalators such as acridine, poly-L-lysine, “end-capping” with one ormore nuclease-resistant linkage groups such as phosphoromonothioate, andthe like.

The preparation of transferrin complexes as carriers of oligonucleotideuptake into cells is described by Wagner et al., Proc. Natl. Acad. Sci.USA 87, 3410-3414 (1990). Cellular delivery of folate-macromoleculeconjugates via folate receptor endocytosis, including delivery of anantisense oligonucleotide, is described by Low et al., U.S. Pat. No.5,108,921. Also see, Leamon et al., Proc. Natl. Acad. Sci. 88, 5572(1991).

The compounds or conjugates of the invention may also be conjugated orfurther conjugated to active drug substances, for example, aspirin,ibuprofen, a sulfa drug, an antidiabetic, an antibacterial agent, achemotherapeutic agent or an antibiotic.

A particularly interesting aspect of the invention is directed to apharmaceutical composition comprising a compound as defined herein or aconjugate as defined herein, and a pharmaceutically acceptable diluent,carrier or adjuvant.

It should be understood that the present invention also particularlyrelevant for a pharmaceutical composition, which comprises a least oneantisense oligonucleotide construct of the invention as an activeingredient. It should be understood that the pharmaceutical compositionaccording to the invention optionally comprises a pharmaceuticalcarrier, and that the pharmaceutical composition optionally comprisesfurther antisense compounds, chemotherapeutic agents, anti-inflammatorycompounds, antiviral compounds and/or immuno-modulating compounds.

As stated, the pharmaceutical composition of the invention may furthercomprise at least one chemotherapeutic agent. The chemotherapeuticcompound is typically selected from the group consisting ofadrenocorticosteroids, such as prednisone, dexamethasone or decadron;altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol);aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole(arimidex); androgens, such as testosterone; asparaginase (elspar);bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane);busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU);chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine,leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine);dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine).

The oligomeric compound or conjugate comprised in this invention can beemployed in a variety of pharmaceutically acceptable salts. As usedherein, the term refers to salts that retain the desired biologicalactivity of the herein identified compounds and exhibit minimalundesired toxicological effects. Non-limiting examples of such salts canbe formed with organic amino acid and base addition salts formed withmetal cations such as zinc, calcium, bismuth, barium, magnesium,aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and thelike, or with a cation formed from ammonia,N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, orethylenediamine; or (c) combinations of (a) and (b); e.g., a zinctannate salt or the like.

In one embodiment of the invention the oligomeric compound of conjugatemay be in the form of a pro-drug. Oligonucleotides are by virtuenegatively charged ions. Due to the lipophilic nature of cell membranesthe cellular uptake of oligonucleotides are reduced compared to neutralor lipophilic equivalents. This polarity “hindrance” can be avoided byusing the pro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S.T. Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140). In this approach the oligonucleotides areprepared in a protected manner so that the oligo is neutral when it isadministered. These protection groups are designed in such a way that sothey can be removed then the oligo is taken up be the cells. Examples ofsuch protection groups are S-acetylthioethyl (SATE) orS-pivaloylthioethyl (t-butyl-SATE). These protection groups are nucleaseresistant and are selectively removed intracellulary.

The invention also includes the formulation of one or moreoligonucleotide compound or conjugate as disclosed herein.Pharmaceutically acceptable binding agents and adjuvants may comprisepart of the formulated drug. Capsules, Tablets and pills etc. maycontain for example the following compounds: microcrystalline cellulose,gum or gelatin as binders; starch or lactose as excipients; stearates aslubricants; various sweetening or flavouring agents. For capsules thedosage unit may contain a liquid carrier like fatty oils. Likewisecoatings of sugar or enteric agents may be part of the dosage unit. Theoligonucleotide formulations may also be emulsions of the activepharmaceutical ingredients and a lipid forming a micellular emulsion.

An oligonucleotide of the invention may be mixed with any material thatdo not impair the desired action, or with material that supplement thedesired action. These could include other drugs including othernucleotide compounds.

For parenteral, subcutaneous, intradermal or topical administration theformulation may include a sterile diluent, buffers, regulators oftonicity and antibacterials. The active compound may be prepared withcarriers that protect against degradation or immediate elimination fromthe body, including implants or microcapsules with controlled releaseproperties. For intravenous administration the preferred carriers arephysiological saline or phosphate buffered saline.

Preferably, an oligomeric compound is included in a unit formulationsuch as in a pharmaceutically accepTable carrier or diluent in an amountsufficient to deliver to a patient a therapeutically effective amountwithout causing serious side effects in the treated patient.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be (a) oral (b) pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, (c) topical including epidermal, transdermal,ophthalmic and to mucous membranes including vaginal and rectaldelivery; or (d) parenteral including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration. Inone embodiment the active oligo is administered IV, IP, orally,topically or as a bolus injection or administered directly in to thetarget organ.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, sprays, suppositories, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Compositionsand formulations for oral administration include but is not restrictedto powders or granules, microparticulates, nanoparticulates, suspensionsor solutions in water or non-aqueous media, capsules, gel capsules,sachets, Tablets or miniTablets. Compositions and formulations forparenteral, intrathecal or intraventricular administration may includesterile aqueous solutions which may also contain buffers, diluents andother suiTable additives such as, but not limited to, penetrationenhancers, carrier compounds and other pharmaceutically accepTablecarriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to tumour tissue may be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(1):3-27).

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, Tablets,capsules, gel capsules, liquid syrups, soft gels and suppositories. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

LNA containing oligomeric compound are useful for a number oftherapeutic applications as indicated above. In general, therapeuticmethods of the invention include administration of a therapeuticallyeffective amount of an LNA-modified oligonucleotide to a mammal,particularly a human.

In a certain embodiment, the present invention provides pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g. mithramycin andoligonucleotide), sequentially (e.g. mithramycin and oligonucleotide fora period of time followed by another agent and oligonucleotide), or incombination with one or more other such chemotherapeutic agents or incombination with radiotherapy. All chemotherapeutic agents known to aperson skilled in the art are here incorporated as combinationtreatments with compound according to the invention.

Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, antiviral drugs, andimmuno-modulating drugs may also be combined in compositions of theinvention. Two or more combined compounds may be used together orsequentially.

In another embodiment, compositions of the invention may contain one ormore antisense compounds, particularly oligonucleotides, targeted to afirst nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Two or more combined compoundsmay be used together or sequentially.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, and the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.

Optimum dosages may vary depending on the relative potency of individualoligonucleotides. Generally it can be estimated based on EC50s found tobe effective in in vitro and in vivo animal models. In general, dosageis from 0.01 μg to 1 g per kg of body weight, and may be given once ormore daily, weekly, monthly or yearly, or even once every 2 to 10 yearsor by continuous infusion for hours up to several months. The repetitionrates for dosing can be estimated based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state.

As stated, in an interesting embodiment of the invention, the oligomericcompounds contain at least one unit of chemistry termed LNA (LockedNucleic Acid).

LNA monomer typically refers to a bicyclic nucleotide analogue whereinthe nucleoside moiety is an analogue as described in the InternationalPatent Application WO 99/14226 and subsequent applications, WO0056746,WO0056748, WO0066604, WO000125248, WO0228875, WO2002094250 andPCT/DK02/00488 all incorporated herein by reference. Preferred LNAnucleotide structures for forming a compound of the invention areexemplified in Scheme 2

wherein X and Y are independently selected among the groups —O—, —S—,—N(H)—, N(R)—, —CH₂— or —CH— (if part of a double bond), —CH₂—O—,—CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if part of adouble bond), —CH═CH—, where R is selected form hydrogen and C₁₋₄-alky;wherein Z and Z* are independently absent, selected among aninternucleoside linkage, a terminal group or a protecting group. Inembodiments wherein at least one of the LNA nucleotides is at the3′-end, at said position Z is a terminal group and Z* is aninternucleoside linkage. In embodiments wherein at least one of the LNAnucleotides is at the 5′-end, at said position Z is absent and Z* is aterminal group. Within the nucleotide sequence, Z is absent and Z* is aninternucleoside linkage. The asymmetric groups may be found in eitherorientation. In Scheme 2, the 4 chiral centers are shown in a fixedconfiguration. However, the configurations in Scheme 2 are notnecessarily fixed. Also comprised in this invention are compounds of thegeneral Scheme 2 in which the chiral centers are found in differentconfigurations, such as those represented in Scheme 3 or 4. Thus, theintention in the illustration of Scheme 2 is not to limit theconfiguration of the chiral centre. Each chiral center in Scheme 2 canexist in either R or S configuration. The definition of R (rectus) and S(sinister) are described in the IUPAC 1974 Recommendations, Section E,Fundamental Stereochemistry: The rules can be found in Pure Appl. Chem.45, 13-30, (1976) and in “Nomenclature of organic Chemistry” pergamon,New York, 1979.

Z and Z* serve for forming an internucleoside linkage, are a terminalgroup or a protecting group, depending on the position of the LNA withinthe compound, namely within the subsequence or at the 3′ end of thesubsequence or compound.

The internucleoside linkage may be —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R^(H))—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—,—O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) isselected form hydrogen and C₁₋₄-alkyl,

The terminal groups are selected independently among from hydrogen,azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto,Prot-S—, Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted C₂₋₆-alkenyloxy, optionally substitutedC₂₋₆-alkynyl, optionally substituted C₂₋₆-alkynyloxy, monophosphate,monothiophosphate, diphosphate, dithiophosphate, triphosphate,trithiophosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl;

The protection groups of hydroxy substituents comprises substitutedtrityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy(MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy(pixyl), optionally substituted methoxytetrahydro-pyranyloxy (mthp),silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS),tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, andphenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxygroups), acyloxy such as acetyl or halogen substituted acetyls, e.g.chloroacetyloxy or fluoroacetyloxy, isobutyryloxy, pivaloyloxy,benzoyloxy and substituted benzoyls, methoxymethyloxy (MOM), benzylethers or substituted benzyl ethers such as 2,6-dichlorobenzyloxy(2,6-Cl₂Bzl). Alternatively when Z or Z* is hydroxyl they may beprotected by attachment to a solid support optionally through a linker.

When Z or Z* is amino groups illustrative examples of the aminoprotection protections are fluorenylmethoxycarbonylamino (Fmoc),tert-butyloxycarbonylamino (BOC), trifluoroacetylamino,allyloxycarbonylamino (alloc, AOC), Z benzyloxycarbonylamino (Cbz),substituted benzyloxycarbonylaminos such as 2-chlorobenzyloxycarbonylamino (2-CIZ), monomethoxytritylamino (MMT),dimethoxytritylamino (DMT), phthaloylamino, and9-(9-phenyl)xanthenylamino (pixyl).

In the embodiment above, Act designates an activation group for —OH,—SH, and —NH(R^(H)), respectively. Such activation groups are, e.g.,selected from optionally substituted O-phosphoramidite, optionallysubstituted O-phosphortriester, optionally substitutedO-phosphordiester, optionally substituted H-phosphonate, and optionallysubstituted O-phosphonate.

In the present context, the term “phosphoramidite” means a group of theformula —P(OR^(x))—N(R^(y))₂, wherein R^(x) designates an optionallysubstituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and eachof R^(y) designate optionally substituted alkyl groups, e.g. ethyl orisopropyl, or the group —N(R^(y))₂ forms a morpholino group(—N(CH₂CH₂)₂O). R^(x) preferably designates 2-cyanoethyl and the twoR^(y) are preferably identical and designate isopropyl. Thus, anespecially relevant phosphoramidite isN,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

B constitutes a natural or non-natural nucleobase and selected amongadenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine,guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil,5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine,2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine,7-propyne-7-deazaguanine, 2-chloro-6-aminopurine.

Particularly preferred bicyclic structures are shown in Scheme 3 below:

Where Y is —O—, —S—, —NH—, or N(R^(H)),

Z and Z* are independently absent, selected among an internucleosidelinkage, a terminal group or a protecting group. In embodiments whereinat least one of the LNA nucleotides is at the 3′-end, at said position Zis a terminal group and Z* is an internucleoside linkage. In embodimentswherein at least one of the LNA nucleotides is at the 5′-end, at saidposition Z is absent and Z* is a terminal group. Within the nucleotidesequence, Z is absent and Z* is an internucleoside linkage.

The internucleotide linkage may be —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected form hydrogen and C₁₋₄-alkyl.

The terminal groups are selected independently among from hydrogen,azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto,Prot-S—, Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, optionally substituted monophosphate,monothiophosphate, diphosphate, dithiophosphate triphosphate,trithiophosphate, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl.

The protection groups of hydroxy substituents comprises substitutedtrityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy(MMT), optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl),optionally substituted methoxytetrahydropyranyloxy (mthp), silyloxy suchas trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS),tert-butyl-dimethylsilyloxy (TBDMS), triethylsilyloxy, andphenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxygroups), acyloxy such as acetyl Alternatively when Z or Z* is hydroxylthey may be protected by attachment to a solid support optionallythrough a linker.

Specifically preferred LNA units are shown in scheme 4.

When Z or Z* is amino groups, illustrative examples of the aminoprotection protections are fluorenylmethoxycarbonylamino (Fmoc),tert-butyloxycarbonylamino (BOC), trifluoroacetylamino,allyloxycarbonylamino (alloc, AOC), monomethoxytritylamino (MMT),dimethoxytritylamino (DMT), phthaloylamino.

In the embodiments above, Act designates an activation group for —OH,—SH, and —NH(R^(H)), respectively. Such activation groups are, e.g.,selected from optionally substituted O-phosphoramidite, optionallysubstituted O-phosphortriester, optionally substitutedO-phosphordiester, optionally substituted H-phosphonate, and optionallysubstituted O-phosphonate.

In the present context, the term “phosphoramidite” means a group of theformula —P(OR^(x))—N(R^(y))₂, wherein R^(x) designates an optionallysubstituted alkyl group, e.g. methyl, 2-cyanoethyl, and each of R^(y)designate optionally substituted alkyl groups, R^(x) preferablydesignates 2-cyanoethyl and the two R^(y) are preferably identical anddesignate isopropyl. Thus, an especially relevant phosphoramidite isN,N-diisopropyl-O-(2-cyanoethyl)-phosphoramidite.

B, in the context of Schemes 3 and 4, constitutes a natural ornon-natural nucleobase and selected among adenine, cytosine,5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine,uracil, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine,inosine, diaminopurine, 2-chloro-6-aminopurine.

A person skilled in the art will appreciate that oligomeric compoundscontaining LNA can be used to combat survivin linked diseases by manydifferent principles, which thus falls within the spirit of the presentinvention.

For instance, LNA oligomeric compounds may be designed as antisenseinhibitors, which are single stranded nucleic acids that prevent theproduction of a disease causing protein, by intervention at the mRNAlevel. Also, they may be designed as Ribozymes or Oligozymes which areantisense oligonucleotides which in addition to the target bindingdomain(s) comprise a catalytic activity that degrades the target mRNA(ribozymes) or comprise an external guide sequence (EGS) that recruit anendogenous enzyme (RNase P) which degrades the target mRNA (oligozymes)

Equally well, the LNA oligomeric compounds may be designed as siRNA'swhich are small double stranded RNA molecules that are used by cells tosilence specific endogenous or exogenous genes by an as yet poorlyunderstood “antisense-like” mechanism.

LNA oligomeric compounds may also be designed as Aptamers (and avariation thereof, termed Spiegelmers) which are nucleic acids thatthrough intra-molecular hydrogen bonding adopt three-dimensionalstructures that enable them to bind to and block their biologicaltargets with high affinity and specificity. Also, LNA oligomericcompounds may be designed as Decoys, which are small double-strandednucleic acids that prevent cellular transcription factors fromtransactivating their target genes by selectively blocking their DNAbinding site.

Furthermore, LNA oligomeric compounds may be designed as Chimeraplasts,which are small single stranded nucleic acids that are able tospecifically pair with and alter a target gene sequence. LNA containingoligomeric compounds exploiting this principle therefore may beparticularly useful for treating survivin linked diseases that arecaused by a mutation in the survivin gene.

Dictated in part by the therapeutic principle by which theoligonucleotide is intended to operate, the LNA oligomeric compounds inaccordance with this invention preferably comprise from about 8 to about60 nucleobases i.e. from about 8 to about 60 linked nucleotides.Particularly preferred compounds are antisense oligonucleotidescomprising from about 12 to about 30 nucleobases and most preferably areantisense compounds comprising about 12-20 nucleobases. The compoundsshown in Table 1 and 2 are all 16-mers.

Referring to the above principles by which an LNA oligomeric compoundcan elicit its therapeutic action the target of the present inventionmay be the survivin gene, the mRNA or the protein. In the most preferredembodiment the LNA oligomeric compounds is designed as an antisenseinhibitor directed against the survivin pre-mRNA or survivin mRNA. Theoligonucleotides may hybridize to any site along the survivin pre-mRNAor mRNA such as sites in the 5′ untranslated leader, exons, introns and3′ untranslated tail.

In a preferred embodiment, the oligonucleotide hybridizes to a portionof the human survivin pre-mRNA or mRNA that comprises thetranslation-initiation site. More preferably, the survivinoligonucleotide comprises a CAT sequence, which is complementary to theAUG initiation sequence of the survivin pre-mRNA or RNA. In anotherembodiment, the survivin oligonucleotide hybridizes to a portion of thesplice donor site of the human survivin pre-mRNA. In yet anotherembodiment, survivin oligonucleotide hybridizes to a portion of thesplice acceptor site of the human survivin pre-mRNA. In anotherembodiment, the survivin oligonucleotide hybridizes to portions of thehuman survivin pre-mRNA or mRNA involved in polyadenylation, transportor degradation.

The skilled person will appreciate that preferred oligonucleotides arethose that hybridize to a portion of the survivin pre-mRNA or mRNA whosesequence does not commonly occur in transcripts from unrelated genes soas to maintain treatment specificity.

The oligomeric compound of the invention are designed to be sufficientlycomplementary to the target to provide the desired clinical responsee.g. the oligomeric compound must bind with sufficient strength andspecificity to its target to give the desired effect. In one embodiment,said compound modulating survivin is designed so as to also modulateother specific nucleic acids which do not encode survivin.

It is preferred that the oligomeric compound according to the inventionis designed so that intra- and intermolecular oligonucleotidehybridisation is avoided.

In many cases the identification of an LNA oligomeric compound effectivein modulating survivin activity in vivo or clinically is based onsequence information on the target gene. However, one of ordinary skillin the art will appreciate that such oligomeric compounds can also beidentified by empirical testing. As such survivin oligomeric compoundshaving, for example, less sequence homology, greater or fewer modifiednucleotides, or longer or shorter lengths, compared to those of thepreferred embodiments, but which nevertheless demonstrate responses inclinical treatments, are also within the scope of the invention.

In one embodiment of the invention the oligomeric compounds are suitableantisense drugs. The design of a potent and safe antisense drug requiresthe fine-tuning of diverse parameters such as affinity/specificity,stability in biological fluids, cellular uptake, mode of action,pharmacokinetic properties and toxicity.

Affinity & specificity: LNA with an oxymethylene 2′-O, 4′-C linkage(β-D-oxy-LNA), exhibits unprecedented binding properties towards DNA andRNA target sequences. Likewise LNA derivatives, such as amino-, thio-and α-L-oxy-LNA display unprecedented affinities towards complementaryRNA and DNA and in the case of thio-LNA the affinity towards RNA is evenbetter than with the β-D-oxy-LNA.

In addition to these remarkable hybridization properties, LNA monomerscan be mixed and act cooperatively with DNA and RNA monomers, and withother nucleic acid analogues, such as 2′-O-alkyl modified RNA monomers.As such, the oligonucleotides of the present invention can be composedentirely of β-D-oxy-LNA monomers or it may be composed of β-D-oxy-LNA inany combination with DNA, RNA or contemporary nucleic acid analogueswhich includes LNA derivatives such as for instance amino-, thio- andα-L-oxy-LNA. The unprecedented binding affinity of LNA towards DNA orRNA target sequences and its ability to mix freely with DNA, RNA and arange of contemporary nucleic acid analogues has a range of importantconsequences according to the invention for the development of effectiveand safe antisense compounds.

Firstly, in one embodiment of the invention it enables a considerableshortening of the usual length of an antisense oligo (from 20-25 mersto, e.g., 12-16 mers) without compromising the affinity required forpharmacological activity. As the intrinsic specificity of an oligo isinversely correlated to its length, such a shortening will significantlyincrease the specificity of the antisense compound towards its RNAtarget. One embodiment of the invention is to, due to the sequence ofthe humane genome is available and the annotation of its genes rapidlyprogressing, identify the shortest possible, unique sequences in thetarget mRNA.

In another embodiment, the use of LNA to reduce the size of oligossignificantly eases the process and prize of manufacture thus providingthe basis for antisense therapy to become a commercially competitivetreatment offer for a diversity of diseases.

In another embodiment, the unprecedented affinity of LNA can be used tosubstantially enhance the ability of an antisense oligo to hybridize toits target mRNA in-vivo thus significantly reducing the time and effortrequired for identifying an active compound as compared to the situationwith other chemistries.

In another embodiment, the unprecedented affinity of LNA is used toenhance the potency of antisense oligonucleotides thus enabling thedevelopment of compounds with more favorable therapeutic windows thanthose currently in clinical trials.

When designed as an antisense inhibitor, the oligonucleotides of theinvention bind to the target nucleic acid and modulate the expression ofits cognate protein. Preferably, such modulation produces an inhibitionof expression of at least 10% or 20% compared to the normal expressionlevel, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, or 90%inhibition compared to the normal expression level.

Typically, the LNA oligonucleotides of the invention will contain otherresidues than β-D-oxy-LNA such as native DNA monomers, RNA monomers,N3′-P5′ phosphoroamidates, 2′-F, 2′-O-Me, 2′-O-methoxyethyl (MOE),2′-O-(3-aminopropyl) (AP), hexitol nucleic acid (HNA), 2′-F-arabinonucleic acid (2′-F-ANA) and D-cyclohexenyl nucleoside (CeNA). Also, theβ-D-oxy-LNA-modified oligonucleotide may also contain other LNA units inaddition to or in place of an oxy-LNA group. In particular, preferredadditional LNA units include thio-LNA or amino-LNA monomers in eitherthe D-β or L-α configurations or combinations thereof or ena-LNA. Ingeneral, an LNA-modified oligonucleotide will contain at least about 5,10, 15 or 20 percent LNA units, based on total nucleotides of theoligonucleotide, more typically at least about 20, 25, 30, 40, 50, 60,70, 80 or 90 percent LNA units, based on total bases of theoligonucleotide.

Stability in biological fluids: One embodiment of the invention includesthe incorporation of LNA monomers into a standard DNA or RNAoligonucleotide to increase the stability of the resulting oligomericcompound in biological fluids e.g. through the increase of resistancetowards nucleases (endonucleases and exonucleases). The extent ofstability will depend on the number of LNA monomers used, their positionin the oligonucleotide and the type of LNA monomer used. Compared to DNAand phosphorothioates the following order of ability to stabilize anoligonucleotide against nucleolytic degradation can be established:DNA<<phosphorothioates˜oxy-LNA<α-L-LNA<amino-LNA<thio-LNA.

Given the fact that LNA is compatible with standard DNA synthesis andmixes freely with many contemporary nucleic acid analogues nucleaseresistance of LNA-oligomeric compounds can be further enhanced accordingto the invention by either incorporating other analogues that displayincreased nuclease stability or by exploiting nuclease-resistantinternucleoside linkages e.g. phosphoromonothioate, phosphorodithioate,and methylphosphonate linkages, etc.

Mode of action: Antisense compounds according to the invention mayelicit their therapeutic action via a variety of mechanisms and may beable to combine several of these in the same compound. In one scenario,binding of the oligonucleotide to its target (pre-mRNA or mRNA) acts toprevent binding of other factors (proteins, other nucleic acids, etc.)needed for the proper function of the target i.e. operate by sterichindrance. For instance, the antisense oligonucleotide may bind tosequence motifs in either the pre-mRNA or mRNA that are important forrecognition and binding of transacting factors involved in splicing,poly-adenylation, cellular transport, post-transcriptional modificationsof nucleosides in the RNA, capping of the 5′-end, translation, etc. Inthe case of pre-mRNA splicing, the outcome of the interaction betweenthe oligonucleotide and its target may be either suppression ofexpression of an undesired protein, generation of alternative splicedmRNA encoding a desired protein or both.

In another embodiment, binding of the oligonucleotide to its targetdisables the translation process by creating a physical block to theribosomal machinery, i.e. translational arrest.

In yet another embodiment, binding of the oligonucleotide to its targetinterferes with the RNAs ability to adopt secondary and higher orderstructures that are important for its proper function, i.e. structuralinterference. For instance, the oligonucleotide may interfere with theformation of stem-loop structures that play crucial roles in differentfunctions, such as providing additional stability to the RNA or adoptingessential recognition motifs for different proteins.

In still another embodiment, binding of the oligonucleotide inactivatesthe target toward further cellular metabolic processes by recruitingcellular enzymes that degrades the mRNA. For instance, theoligonucleotide may comprise a segment of nucleosides that have theability to recruit ribonuclease H (RNaseH) that degrades the RNA part ofa DNA/RNA duplex. Likewise, the oligonucleotide may comprise a segmentwhich recruits double stranded RNAses, such as for instance RNAseIII orit may comprise an external guide sequence (EGS) that recruit anendogenous enzyme (RNase P) which degrades the target mRNA Also, theoligonucleotide may comprise a sequence motif which exhibit RNAsecatalytic activity or moieties may be attached to the oligonucleotideswhich when brought into proximity with the target by the hybridizationevent disables the target from further metabolic activities.

It has been shown that β-D-oxy-LNA does not support RNaseH activity.However, this can be changed according to the invention by creatingchimeric oligonucleotides composed of β-D-oxy-LNA and DNA, calledgapmers. A gapmer is based on a central stretch of 4-12 nt DNA ormodified monomers recognizable and cleavable by the RNaseH (the gap)typically flanked by 1 to 6 residues of β-D-oxy-LNA (the flanks). Theflanks can also be constructed with LNA derivatives. There are otherchimeric constructs according to the invention that are able to act viaan RNaseH mediated mechanism. A headmer is defined by a contiguousstretch of β-D-oxy-LNA or LNA derivatives at the 5′-end followed by acontiguous stretch of DNA or modified monomers recognizable andcleavable by the RNaseH towards the 3′-end, and a tailmer is defined bya contiguous stretch of DNA or modified monomers recognizable andcleavable by the RNaseH at the 5′-end followed by a contiguous stretchof β-D-oxy-LNA or LNA derivatives towards the 3′-end. Other chimerasaccording to the invention, called mixmers consisting of an alternatecomposition of DNA or modified monomers recognizable and cleavable byRNaseH and β-D-oxy-LNA and/or LNA derivatives might also be able tomediate RNaseH binding and cleavage. Since α-L-LNA recruits RNaseHactivity to a certain extent, smaller gaps of DNA or modified monomersrecognizable and cleavable by the RNaseH for the gapmer construct mightbe required, and more flexibility in the mixmer construction might beintroduced. FIG. 1 shows an outline of different designs according tothe invention.

The clinical effectiveness of antisense oligonucleotides depends to asignificant extent on their pharmacokinetics e.g. absorption,distribution, cellular uptake, metabolism and excretion. In turn theseparameters are guided significantly by the underlying chemistry and thesize and three-dimensional structure of the oligonucleotide.

As mentioned earlier LNA according to the invention is not a single, butseveral related chemistries, which although molecularly different allexhibit stunning affinity towards complementary DNA and RNA, Thus, theLNA family of chemistries are uniquely suited of development oligosaccording to the invention with tailored pharmacokinetic propertiesexploiting either the high affinity of LNA to modulate the size of theactive compounds or exploiting different LNA chemistries to modulate theexact molecular composition of the active compounds. In the latter case,the use of for instance amino-LNA rather than oxy-LNA will change theoverall charge of the oligo and affect uptake and distribution behavior.Likewise the use of thio-LNA instead of oxy-LNA will increase thelipophilicity of the oligonucleotide and thus influence its ability topass through lipophilic barriers such as for instance the cell membrane.

Modulating the pharmacokinetic properties of an LNA oligonucleotideaccording to the invention may further be achieved through attachment ofa variety of different moieties. For instance, the ability ofoligonucleotides to pass the cell membrane may be enhanced by attachingfor instance lipid moieties such as a cholesterol moiety, a thioether,an aliphatic chain, a phospholipid or a polyamine to theoligonucleotide. Likewise, uptake of LNA oligonucleotides into cells maybe enhanced by conjugating moieties to the oligonucleotide thatinteracts with molecules in the membrane, which mediates transport intothe cytoplasm.

The pharmacodynamic properties can according to the invention beenhanced with groups that improve oligomer uptake, enhance biostabilitysuch as enhance oligomer resistance to degradation, and/or increase thespecificity and affinity of oligonucleotides hybridisationcharacteristics with target sequence e.g. a mRNA sequence.

There are basically two types of toxicity associated with antisenseoligos: sequence-dependant toxicity, involving the base sequence, andsequence-independent, class-related toxicity. With the exception of theissues related to immunostimulation by native CpG sequence motifs, thetoxicities that have been the most prominent in the development ofantisense oligonucleotides are independent of the sequence, e.g. relatedto the chemistry of the oligonucleotide and dose, mode, frequency andduration of administration. The phosphorothioates class ofoligonucleotides have been particularly well characterized and found toelicit a number of adverse effects such as complement activation,prolonged PTT (partial thromboplastin time), thrombocytopenia,hepatotoxicity (elevation of liver enzymes), cardiotoxicity,splenomegaly and hyperplasia of reticuloendothelial cells.

As mentioned earlier, the LNA family of chemistries provideunprecedented affinity, very high bio-stability and the ability tomodulate the exact molecular composition of the oligonucleotide. In oneembodiment of the invention, LNA containing compounds enables thedevelopment of oligonucleotides which combine high potency withlittle—if any—phosphorothioate linkages and which are therefore likelyto display better efficacy and safety than contemporary antisensecompounds.

Oligo- and polynucleotides of the invention may be produced using thepolymerisation techniques of nucleic acid chemistry well known to aperson of ordinary skill in the art of organic chemistry. Generally,standard oligomerisation cycles of the phosphoramidite approach (S. L.Beaucage and R. P. Iyer, Tetrahedron, 1993, 49, 6123; S. L. Beaucage andR. P. Iyer, Tetrahedron, 1992, 48, 2223) is used, but e.g. H-phosphonatechemistry, phosphortriester chemistry can also be used.

For some monomers of the invention longer coupling time, and/or repeatedcouplings with fresh reagents, and/or use of more concentrated couplingreagents were used. The phosphoramidites employed coupled withsatisfactory >95% step-wise coupling yields. Thiolation of the phosphateis performed by exchanging the normal, e.g. iodine/pyridine/H₂O,oxidation used for synthesis of phosphordiester oligomers with anoxidation using Beaucage's reagent (commercially available) othersulfurisation reagents are also comprised. The phosphorthioate LNAoligomers were efficiently synthesised with stepwise coupling yields>=98%.

The β-D-amino-LNA, β-D-thio-LNA oligonucleotides, α-L-LNA andβ-D-methylamino-LNA oligonucleotides were also efficiently synthesisedwith step-wise coupling yields ≧98% using the phosphoramiditeprocedures.

Purification of LNA oligomeric compounds was done using disposablereversed phase purification cartridges and/or reversed phase HPLC and/orprecipitation from ethanol or butanol. Capillary gel electrophoresis,reversed phase HPLC, MALDI-MS, and ESI-MS was used to verify the purityof the synthesized oligonucleotides. Furthermore, solid supportmaterials having immobilised thereto an optionally nucleobase protectedand optionally 5′-OH protected LNA are especially interesting asmaterial for the synthesis of LNA containing oligomeric compounds wherean LNA monomer is included in at the 3′ end. In this instance, the solidsupport material is preferable CPG, e.g. a readily (commercially)available CPG material or polystyrene onto which a 3′-functionalised,optionally nucleobase protected and optionally 5′-OH protected LNA islinked using the conditions stated by the supplier for that particularmaterial.

As it must be clear by now, an interesting aspect of the invention isdirected to a compound of the invention or a conjugate of the inventionfor use as a medicament. As it must also be unambiguous by now, the useof a compound of the invention or as conjugate of the invention for themanufacture of a medicament for the treatment of cancer is aparticularly interesting aspect of the invention.

The pharmaceutical composition according to the invention can be usedfor the treatment of many different diseases. For example survivin hasbeen found to be overexpressed in human tumours of lung (Monzo et al.,1999, J. Clin. Oncol 17, 2100-2104), breast (Tanaka et al., 2000, Clin.Cancer Res. 6, 127-134; Nasu et al., 2002, Anticancer Res. 22,1839-1844), colon/rectum (Kawasaki et al., 1998, Cancer Res. 58,5071-5074; Rbdel et al., 2002, Strahlenther. Onkol. 8, 426-434), stomach(Lu et al., 1998, Cancer Res. 58, 1808-1812; Tsuburaya et al., 2002,Hepatogastroenterology 49, 1150-1152), oesophagus (Kato et al., 2001,Int. J. Cancer 95, 92-95; Ikeguchi and Kaibara, 2002, Br. J. Cancer 87,883-887), pancreas (Satoh et al., 2001, Cancer 92, 271-278; Sarela etal., 2002, Br. J. Cancer 86, 886-892), liver (Ikeguchi et al., 2002,Clin. Cancer Res. 8, 3131-3136), uterus (Saitoh et al., 1999, Int. J.Oncol. 15, 137-141; Takai et al., 2002, Cancer Lett. 184, 105-116),ovaries (Yoshoda et al., 2001, Int. J. Oncol. 19, 537-542; Takai et al.,2002, Int. J. Mol. Med. 10, 211-216), Hodgkin's disease (Garcia et al.,2003, Blood 101, 681-689), non-Hodgkin's lymphoma (Adida et al., 2000,Blood 96, 1921-1925; Kuttler et al., 2002, Leukemia 16, 726-735),leukemias (Adida et al., 2000, Br. J. Haematol. 111, 196-203; Kamihiraet al., 2001, Br. J. Haematol. 114, 63-69; Mori et al., 2001, Int. J.Haematol. 75, 161-165), neuroblastoma (Islam et al., 2000, Oncogene 19,617-623; Adida et al., 1998, Lancet 351, 882-883), phaeochromocytoma(Koch et al., 2002, Eur. J. Endocrinol. 146, 381-388), soft tissuesarcomas (Würl et al., 2002, Lancet 359, 943-945), gliomas (Chakravartiet al. 2002, J. Clin. Oncol. 20, 1063-1068), melanoma (Grossman et al.,1999, J. Invest. Dermatol. 113, 1076-1081), bladder (Swana et al., 1999,New Engl. J. Med. 341, 452-453; Smith et al., 2001, JAMA 285, 324-328),cervix (Kim et al., 2002, Anticancer Res. 22, 805-808; Yoshida et al.,2003, Oncol. Rep. 10, 45-49), prostate (Ambrosini et al., 1997, Nat.Med. 3, 917-921). Like cancer cells proliferating vascular endothelialcells are sensitive to downregulation of survivin expression. Thepharmaceutical composition according to the invention can therefore beused in the treatments of diseases characterized by abnormal diseasecausing angiogenesis. Examples of such diseases are cancers in generaland artherosclerosis, psoriasis, diabetic retinopathy, rheumatoidarthritis, asthma, warts, allergic dermatitis and Karposis sarcoma.Furthermore, survivin may be actively involved in regulating cellviability during HIV-1 infection (Zhu et al., 2003, Apoptosis 8, 71-79).Survivin is essential to the correct execution of mitosis and completionof cell division. Downregulation of survivin should therefore berelevant in the treatment of any disease characterized by uncontrolledor abnormal cell growth.

Generally stated, one aspect of the invention is directed to a method oftreating a mammal suffering from or susceptible to a disease caused byabnormal angiogenesis, comprising administering to the mammal antherapeutically effective amount of an oligonucleotide targeted tosurvivin that comprises one or more LNA units.

An interesting aspect of the invention is directed to the use of acompound as defined herein or as conjugate as defined herein for thepreparation of a medicament for the treatment of artherosclerosis,psoriasis, diabetic retinopathy, rheumatoid arthritis, asthma, warts andallergic dermatitis.

The methods of the invention is preferably employed for treatment orprophylaxis against diseases caused by cancer, particularly fortreatment of cancer as may occur in tissue such as lung, breast, colon,prostate, pancreas, liver, brain, testes, stomach, intestine, bowel,spinal cord, sinuses, urinary tract or ovaries cancer.

Furthermore, the invention described herein encompasses a method ofpreventing or treating cancer comprising a therapeutically effectiveamount of a survivin modulating oligomeric compound, including but notlimited to high doses of the oligomer, to a human in need of suchtherapy. The invention further encompasses the use of a short period ofadministration of a survivin modulating oligomeric compound. Normal,non-cancerous cells divide at a frequency characteristic for theparticular cell type. When a cell has been transformed into a cancerousstate, uncontrolled cell proliferation and reduced cell death results,and therefore, promiscuous cell division or cell growth is a hallmark ofa cancerous cell type. Examples of types of cancer, include, but are notlimited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g.,acute leukemia such as acute lymphocytic leukemia, acute myelocyticleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia,multiple myeloma), colon carcinoma, rectal carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer,testicular cancer, lung carcinoma, bladder carcinoma, melanoma, head andneck cancer, brain cancer, cancers of unknown primary site, neoplasms,cancers of the peripheral nervous system, cancers of the central nervoussystem, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, small cell lung carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma), heavychain disease, metastases, or any disease or disorder characterized byuncontrolled or abnormal cell growth.

In the use of a compound of the invention or as conjugate of theinvention for the manufacture of a medicament for the treatment ofcancer, said cancer may suitably be in the form of a solid tumor.Furthermore, said cancer is also suitably a carcinoma. The carcinoma istypically in the from selected from the group consisting of malignantmelanoma, basal cell carcinoma, ovarian carcinoma, breast carcinoma,non-small cell lung cancer, renal cell carcinoma, bladder carcinoma,recurrent superficial bladder cancer, stomach carcinoma, prostaticcarcinoma, pancreatic carcinoma, lung carcinoma, cervical carcinoma,cervical dysplasia, laryngeal papillomatosis, colon carcinoma,colorectal carcinoma and carcinoid tumors. More typically, saidcarcinoma is selected from the group consisting of malignant melanoma,non-small cell lung cancer, breast carcinoma, colon carcinoma and renalcell carcinoma. The malignant melanoma is typically selected from thegroup consisting of superficial spreading melanoma, nodular melanoma,lentigo maligna melanoma, acral melagnoma, amelanotic melanoma anddesmoplastic melanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma istypically in the form selected from the group consisting ofosteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibroushistiocytoma, fibrosarcoma and Kaposi's sarcoma.

Alternatively, the cancer may suitably be a glioma.

It should be understood that the invention also relates to apharmaceutical composition, which comprises a least one antisenseoligonucleotide construct of the invention as an active ingredient. Itshould be understood that the pharmaceutical composition according tothe invention optionally comprises a pharmaceutical carrier, and thatthe pharmaceutical composition optionally comprises further antisensecompounds, chemotherapeutic compounds, anti-inflammatory compounds,antiviral compounds and/or immuno-modulating compounds.

The oligomeric compound comprised in this invention can be employed in avariety of pharmaceutically acceptable salts. As used herein, the termrefers to salts that retain the desired biological activity of theherein identified compounds and exhibit minimal undesired toxicologicaleffects. Non-limiting examples of such salts can be formed with organicamino acid and base addition salts formed with metal cations such aszinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt,nickel, cadmium, sodium, potassium, and the like, or with a cationformed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

In one embodiment of the invention the oligomeric compound may be in theform of a pro-drug. Oligonucleotides are by virtue negatively chargedions. Due to the lipophilic nature of cell membranes the cellular uptakeof oligonucleotides are reduced compared to neutral or lipophilicequivalents. This polarity “hindrance” can be avoided by using thepro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T.Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140). In this approach the oligonucleotides areprepared in a protected manner so that the oligo is neutral when it isadministered. These protection groups are designed in such a way that sothey can be removed then the oligo is taken up be the cells. Examples ofsuch protection groups are S-acetylthioethyl (SATE) orS-pivaloylthioethyl (t-butyl-SATE). These protection groups are nucleaseresistant and are selectively removed intracellulary.

In one embodiment of the invention the oligomeric compound is linked toligands/conjugates. It is way to increase the cellular uptake ofantisense oligonucleotides. This conjugation can take place at theterminal positions 5′/3′-OH but the ligands may also take place at thesugars and/or the bases. Other examples of conjugates/lingands arecholesterol moieties, duplex intercalators such as acridine,poly-L-lysine, “end-capping” with one or more nuclease-resistant linkagegroups.

The invention also includes the formulation of one or moreoligonucleotide compound as disclosed herein. Pharmaceuticallyacceptable binding agents and adjuvants may comprise part of theformulated drug. Capsules, tablets and pills etc. may contain forexample the following compounds: microcrystalline cellulose, gum orgelatin as binders; starch or lactose as excipients; stearates aslubricants; various sweetening or flavouring agents. For capsules thedosage unit may contain a liquid carrier like fatty oils. Likewisecoatings of sugar or enteric agents may be part of the dosage unit. Theoligonucleotide formulations may also be emulsions of the activepharmaceutical ingredients and a lipid forming a micellular emulsion. Anoligonucleotide of the invention may be mixed with any material that donot impair the desired action, or with material that supplement thedesired action. These could include other drugs including othernucleotide compounds. For parenteral, subcutaneous, intradermal ortopical administration the formulation may include a sterile diluent,buffers, regulators of tonicity and antibacterials. The active compoundmay be prepared with carriers that protect against degradation orimmediate elimination from the body, including implants or microcapsuleswith controlled release properties. For intravenous administration thepreferred carriers are physiological saline or phosphate bufferedsaline. Preferably, an oligomeric compound is included in a unitformulation such as in a pharmaceutically acceptable carrier or diluentin an amount sufficient to deliver to a patient a therapeuticallyeffective amount without causing serious side effects in the treatedpatient.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be (a) oral (b) pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, (c) topical including epidermal, transdermal,ophthalmic and to mucous membranes including vaginal and rectaldelivery; or (d) parenteral including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration. Inone embodiment the active oligo is administered IV, IP, orally,topically or as a bolus injection or administered directly in to thetarget organ. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, sprays, suppositories, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Compositions and formulations for oral administration include but is notrestricted to powders or granules, microparticulates, nanoparticulates,suspensions or solutions in water or non-aqueous media, capsules, gelcapsules, sachets, tablets or minitablets. Compositions and formulationsfor parenteral, intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to tumour tissue may be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(1):3-27). The pharmaceutical formulations of the presentinvention, which may conveniently be presented in unit dosage form, maybe prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product. The compositions ofthe present invention may be formulated into any of many possible dosageforms such as, but not limited to, tablets, capsules, gel capsules,liquid syrups, soft gels and suppositories. The compositions of thepresent invention may also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers. Oligomeric compounds of theinvention may also be conjugated to active drug substances, for example,aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial oran antibiotic.

LNA containing oligomeric compound are useful for a number oftherapeutic applications as indicated above. In general, therapeuticmethods of the invention include administration of a therapeuticallyeffective amount of an LNA-modified oligonucleotide to a mammal,particularly a human. In a certain embodiment, the present inventionprovides pharmaceutical compositions containing (a) one or moreantisense compounds and (b) one or more other chemotherapeutic agentswhich function by a non-antisense mechanism. When used with thecompounds of the invention, such chemotherapeutic agents may be usedindividually (e.g. mithramycin and oligonucleotide), sequentially (e.g.mithramycin and oligonucleotide for a period of time followed by anotheragent and oligonucleotide), or in combination with one or more othersuch chemotherapeutic agents or in combination with radiotherapy. Allchemotherapeutic agents known to a person skilled in the art are hereincorporated as combination treatments with compound according to theinvention. Anti-inflammatory drugs, including but not limited tononsteroidal anti-inflammatory drugs and corticosteroids, antiviraldrugs, and immuno-modulating drugs may also be combined in compositionsof the invention. Two or more combined compounds may be used together orsequentially.

Accordingly, a further aspect of the invention is directed to the use ofa compound as defined herein or as conjugate as defined herein for themanufacture of a medicament for the treatment of cancer, wherein saidmedicament further comprises a chemotherapeutic agent selected from thegroup consisting of adrenocorticosteroids, such as prednisone,dexamethasone or decadron; altretamine (hexalen, hexamethylmelamine(HMM)); amifostine (ethyol); aminoglutethimide (cytadren); amsacrine(M-AMSA); anastrozole (arimidex); androgens, such as testosterone;asparaginase (elspar); bacillus calmette-gurin; bicalutamide (casodex);bleomycin (blenoxane); busulfan (myleran); carboplatin (paraplatin);carmustine (BCNU, BiCNU); chlorambucil (leukeran); chlorodeoxyadenosine(2-CDA, cladribine, leustatin); cisplatin (platinol); cytosinearabinoside (cytarabine); dacarbazine (DTIC); dactinomycin(actinomycin-D, cosmegen); daunorubicin (cerubidine); docetaxel(taxotere); doxorubicin (adriomycin); epirubicin; estramustine (emcyt);estrogens, such as diethylstilbestrol (DES); etopside (VP-16, VePesid,etopophos); fludarabine (fludara); flutamide (eulexin); 5-FUDR(floxuridine); 5-fluorouracil (5-FU); gemcitabine (gemzar); goserelin(zodalex); herceptin (trastuzumab); hydroxyurea (hydrea); idarubicin(idamycin); ifosfamide; IL-2 (proleukin, aldesleukin); interferon alpha(intron A, roferon A); irinotecan (camptosar); leuprolide (lupron);levamisole (ergamisole); lomustine (CCNU); mechlorathamine (mustargen,nitrogen mustard); melphalan (alkeran); mercaptopurine (purinethol,6-MP); methotrexate (mexate); mitomycin-C (mutamucin); mitoxantrone(novantrone); octreotide (sandostatin); pentostatin (2-deoxycoformycin,nipent); plicamycin (mithramycin, mithracin); prorocarbazine (matulane);streptozocin; tamoxifin (nolvadex); taxol (paclitaxel); teniposide(vumon, VM-26); thiotepa; topotecan (hycamtin); tretinoin (vesanoid,all-trans retinoic acid); vinblastine (valban); vincristine (oncovin)and vinorelbine (navelbine). Suitably, the further chemotherapeuticagent is selected from taxanes such as Taxol, Paclitaxel or Docetaxel.

Similarly, the invention is further directed to the use of a compound asdefined herein or as conjugate as defined herein for the manufacture ofa medicament for the treatment of cancer, wherein said treatment furthercomprises the administration of a further chemotherapeutic agentselected from the group consisting of adrenocorticosteroids, such asprednisone, dexamethasone or decadron; altretamine (hexalen,hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide(cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, suchas testosterone; asparaginase (elspar); bacillus calmette-gurin;bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran);carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil(leukeran); chlorodeoxyadenosine (2-CDA, cladribine, leustatin);cisplatin (platinol); cytosine arabinoside (cytarabine); dacarbazine(DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, said treatment further comprises theadministration of a further chemotherapeutic agent selected fromtaxanes, such as Taxol, Paclitaxel or Docetaxel.

Alternatively stated, the invention is furthermore directed to a methodfor treating cancer, said method comprising administering a compound asdefined herein, or a conjugate as defined herein or a pharmaceuticalcomposition as defined herein to a patient in need thereof and furthercomprising the administration of a further chemotherapeutic agent. Saidfurther administration may be such that the further chemotherapeuticagent is conjugated to the compound of the invention, is present in thepharmaceutical composition, or is administered in a separateformulation.

In another embodiment, compositions of the invention may contain one ormore antisense compounds, particularly oligonucleotides, targeted to afirst nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Two or more combined compoundsmay be used together or sequentially.

In a preferred embodiment the present invention provides pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which stabilize microtubules ordampen microtubule dynamic—and thereby prevent tension forming at thekinetochores of sister chromatids. Such chemotherapeutic agents includestaxanes, in particular Taxol, Paclitaxel and Docetaxel. When used withthe compounds of the invention, such chemotherapeutic agents should beused sequentially initiating with oligonucleotide treatment for a periodof time which sensitises the target cells to subsequent co-treatmentwith the chemotherapeutic agent by reducing the level of survivinprotein in tumor cells and proliferating endothelial cells of the tumorvasculature.

In another preferred embodiment the present invention providespharmaceutical compositions containing (a) one or more antisensecompounds and (b) radiation therapy. When used with the compounds of theinvention, radiation therapy should be used sequentially initiating witholigonucleotide treatment for a period of time which sensitises thetarget cells to subsequent additional radiotherapy by reducing the levelof survivin protein in tumor cells and proliferating endothelial cellsof the tumor vasculature.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, and the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Optimum dosages may vary depending on the relative potency of individualoligonucleotides.

Generally it can be estimated based on EC50s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to 1g per kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 10 years or by continuousinfusion for hours up to several months. The repetition rates for dosingcan be estimated based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state.

The LNA containing oligomeric compounds of the present invention canalso be utilized for as research reagents for diagnostics, therapeuticsand prophylaxis. In research, the antisense oligonucleotides may be usedto specifically inhibit the synthesis of survivin genes in cells andexperimental animals thereby facilitating functional analysis of thetarget or an appraisal of its usefulness as a target for therapeuticintervention. In diagnostics the antisense oligonucleotides may be usedto detect and quantitate survivin expression in cell and tissues byNorthern blotting, in-situ hybridisation or similar techniques. Fortherapeutics, an animal or a human, suspected of having a disease ordisorder, which can be treated by modulating the expression of survivinis treated by administering antisense compounds in accordance with thisinvention. Further provided are methods of treating an animal particularmouse and rat and treating a human, suspected of having or being proneto a disease or condition, associated with expression of survivin byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

A further aspect of the invention is directed to a method of preventingor limiting apoptosis comprising the administration of a compound asherein, a conjugate as defined herein or a pharmaceutical composition asdefined herein. The prevention of apoptosis may be in vitro or in vivo.The prevention may be done on a cellular assay or within a tissue sampleor within the living mammal.

A related aspect of the invention is directed method of preventingcellular proliferation comprising the administration of a compound asdefined herein, a conjugate as defined herein or a pharmaceuticalcomposition as defined herein. The prevention of proliferation may be invitro or in vivo. The prevention may be done on a cellular assay orwithin a tissue sample or within the living mammal.

The invention is further illustrated in a non-limiting manner by thefollowing examples.

EXAMPLES Example 1 Monomer Synthesis

The LNA monomer building blocks and derivatives thereof were preparedfollowing published procedures and references cited therein, see:

-   WO 03/095467 A1-   D. S. Pedersen, C. Rosenbohm, T. Koch (2002) Preparation of LNA    Phosphoramidites, Synthesis 6, 802-808.-   M. D. Sørensen, L. Kværnø, T. Bryld, A. E. Håkansson, B.    Verbeure, G. Gaubert, P. Herdewijn, J. Wengel (2002)    α-L-ribo-configured Locked Nucleic Acid (α-l-LNA): Synthesis and    Properties, J. Am. Chem. Soc., 124, 2164-2176.-   S. K. Singh, R. Kumar, J. Wengel (1998) Synthesis of Novel    Bicyclo[2.2.1] Ribonucleosides: 2′-Amino- and 2′-Thio-LNA Monomeric    Nucleosides, J. Org. Chem. 1998, 63, 6078-6079.-   C. Rosenbohm, S. M. Christensen, M. D. Sørensen, D. S.    Pedersen, L. E. Larsen, J. Wengel, T. Koch (2003) Synthesis of    2′-amino-LNA: a new strategy, Org. Biomol. Chem. 1, 655-663.

Synthesis of the 2′-thio-LNA ribothymidine phosphoramidite. Reagents andconditions: i) Pd/C, H₂, acetone, MeOH; ii) BzCl, pyridine, DMF; iii)0.25 M H₂SO₄ (aq), DMF, 80° C. (79% from 4; 3 steps); iv) Tf₂O, DMAP,CH₂Cl₂, 0° C.; v) Na₂S, DMF (72% from 7; 2 steps); vi) NaOBz, DMF, 100°C. (81%); vii) NH₃, MeOH (76%); viii) DMT-Cl, pyridine (88%); ix)P(OCH₂CH₂CN)(N(^(i)Pr)₂)₂, 4,5-dicyanoimidazole, CH₂Cl₂ (99%).DMT=4,4′-dimethoxytrityl,PN₂=2-cyanoethoxy(diisopropylamino)phosphinoyl.

1-(3-O-Benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine(7, FIG. 4)

Anhydro-nucleoside 4 (C. Rosenbohm, S. M. Christensen, M. D. Sørensen,D. S. Pedersen, L. E. Larsen, J. Wengel, T. Koch (2003) Synthesis of2′-amino-LNA: a new strategy, Org. Biomol. Chem. 1, 655-663) (30.0 g,58.1 mmol) was heated to 70° C. in a mixture of methanol (1000 cm³) andacetone (1000 cm³) until a clear solution was obtained and the solutionwas allowed to reach room temperature. The reaction flask was flushedwith argon and Pd/C (10 wt. % Pd on carbon, 6.2 g, 5.8 mmol) was added.The mixture was stirred vigorously under an atmosphere of hydrogen gas(balloon). After 23 h the slurry was filtered through a pad of celite.The catalyst was recovered from the celite and refluxed in DMF (1000cm³) for 1 h. The hot DMF slurry was filtered through a pad of celiteand the organic layers combined and evaporated in vacuo to givenucleoside 5 as a yellow powder. Residual solvents were removed on ahigh vacuum pump overnight.

The crude nucleoside 5 (23 g) was heated to 70° C. in DMF (300 cm³) togive a clear yellow solution that was allowed to cool to roomtemperature. Benzoyl chloride (81.7 g, 581 mmol, 67.4 cm³) was addedfollowed by pyridine (70 cm³). After 18 h the reaction was quenched withmethanol (200 cm³) and excess methanol was removed in vacuo. To the darkbrown solution of nucleoside 6 aqueous H₂SO₄ (0.25 M, 400 cm³) wasadded. The solution was heated to 80° C. on an oil bath (At approx 50°C. precipitation occurs. The solution becomes clear again at 80° C.).After 22 h at 80° C. the solution was allowed to cool to roomtemperature. The reaction mixture was transferred to a separatory funnelwith ethyl acetate (1000 cm³). The organic layer was washed with sat. aqNaHCO₃ (2×1000 cm³). The combined aqueous layers were extracted withethyl acetate (1000+500 cm³). The organic layers were combined andwashed with sat. aq NaHCO₃ (1000 cm³), dried (Na₂SO₄), filtered andevaporated in vacuo to give a yellow liquid. Residual solvents wereremoved on a high vacuum pump overnight to give a yellow syrup. Theproduct was purified by Dry Column Vacuum Chromatography (id 10 cm; 100cm³ fractions; 50-100% EtOAc in n-heptane (v/v)—10% increments; 2-24%MeOH in EtOAc (v/v)—2% increments). Fractions containing the productwere combined and evaporated in vacuo giving nucleoside 7 (25.1 g, 79%)as a white foam.

R_(f)=0.54 (5% MeOH in EtOAc, v/v);

ESI-MS m/z found 549.0 ([MH]⁺, calcd 549.1);

¹H NMR (DMSO-d₆) δ 11.39 (br s, 1H, NH), 8.10-8.08 (m, 2H, Ph),7.74-7.70 (m, 1H, Ph), 7.60-7.56 (m, 2H, Ph), 7.51 (d, J=1.1 Hz, 1H,H6), 6.35 (d, J=4.9 Hz, 1H, H1′), 6.32 (d, J=5.3 Hz, 1H, 2′-OH), 5.61(d, J=4.0 Hz, 1H, H3′), 4.69 (d, J=10.8 Hz, 1H), 4.59 (m, 1H, H2′), 4.55(d, J=10.8 Hz, 1H), 4.52 (d, J=10.8 Hz, 1H), 4.46 (d, J=10.6 Hz, 1H)(H₅′ and H1″), 3.28 (s, 3H, Ms), 3.23 (s, 3H, Ms), 1.81 (s, 3H, CH₃);

¹³C NMR (DMSO-d₆) δ 164.5, 163.6 (C4, PhC(O)), 150.3 (C2), 137.7 (C6),133.8, 129.6, 128.7, 128.6 (Ph), 108.1 (C5), 84.8 (C1′), 81.1 (C4′),78.0 (C3′), 73.2 (C2′), 68.0, 67.1 (C5′, C1″), 36.7, 36.6 (2×Ms), 11.9(CH₃);

Elemental anal. calcd for C₂₀H₂₄N₂O₁₂S₂.0.33H₂O (%): C, 44.34; H, 4.65;N, 4.85. Found: C, 44.32; H, 4.58; N, 4.77.

(1R,3R,4R,7R)-7-Benzoyloxy-1-methansulfonyloxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(9)

1-(3-O-Benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine(7) (10.00 g, 18.23 mmol) was dissolved in dichloromethane (500 cm³) andcooled to 0° C. Pyridine (15 cm³) and DMAP (8.91 g, 72.9 mmol) was addedfollowed by dropwise addition of trifluoromethanesulfonic anhydride(10.30 g, 36.5 mmol, 6.0 cm³). After 1 h the reaction was quenched withsat. aq NaHCO₃ (500 cm³) and transferred to a separatory funnel. Theorganic layer was washed with 1.0 M aq HCl (500 cm³), sat. aq NaHCO₃(500 cm³) and brine (500 cm³). The organic layer was evaporated in vacuowith toluene (100 cm³) to give1-(3-O-benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-2-O-trifluoromethanesulfonyl-β-D-threo-pentofuranosyl)thymine(8) as a yellow powder.

The crude nucleoside 8 was dissolved in DMF (250 cm³) and Na₂S (1.57 g,20.1 mmol) was added to give a dark green slurry. After 3 h the reactionwas quenched with half sat. aq NaHCO₃ (500 cm³) and extracted withdichloromethane (500+2×250 cm³). The combined organic layers were washedwith brine (500 cm³), dried (Na₂SO₄), filtered and concentrated in vacuoto give a yellow liquid. Residual solvent was removed overnight on ahigh vacuum pump to give a yellow gum that was purified by Dry ColumnVacuum Chromatography (id 6 cm: 50 cm³ fractions; 50-100% EtOAc inn-heptane (v/v)—10% increments; 2-20% MeOH in EtOAc (v/v)—2% increments)to give nucleoside 9 (6.15 g, 72%) as a yellow foam.

R_(f)=0.27 (20% n-heptane in EtOAc, v/v);

ESI-MS m/z found 469.0 ([MH]⁺, calcd 469.1);

¹H NMR (CDCl₃) δ 58.70 (br s, 1H, NH), 8.01-7.99 (m, 2H, Ph), 7.67 (d,J=1.1 Hz, 1H, H6), 7.65-7.61 (m, 1H, Ph), 7.50-7.46 (m, 2H, Ph), 5.98(s, 1H, H1′), 5.34 (d, J=2.4 Hz, 1H, H3′), 4.66 (d, J=11.7 Hz, 1H,H5′a), 4.53 (d, J=11.5 Hz, 1H, H5′b), 4.12 (m (overlapping with residualEtOAc), 1H, H2′), 3.15-3.13 (m, 4H, H1″a and Ms), 3.06 (d, J=10.6 Hz,1H, H1″b), 1.98 (d, J=1.1 Hz, 3H, CH₃);

¹³C NMR (CDCl₃) δ 165.2, 163.5 (C4, PhC(O)), 149.9 (C2), 134.1, 133.9,129.8, 128.7, 128.3 (C6, Ph), 110.7 (C5), 91.1 (C1′), 86.8 (C4′), 72.6(C3′), 65.8 (C5′), 50.5 (C2′), 37.9 (Ms), 35.1 (C1″), 12.5 (CH₃);

Elemental anal. calcd for C₁₉H₂₀N₂O₈S₂.0.33 EtOAc (%): C, 49.21; H,4.72; N, 5.47. Found: C, 49.25; H, 4.64; N, 5.48.

(1R,3R,4R,7R)-7-Benzoyloxy-1-benzoyloxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(10)

Nucleoside 9 (1.92 g, 4.1 mmol) was dissolved in DMF (110 cm³). Sodiumbenzoate (1.2 g, 8.2 mmol) was added and the mixture was heated to 100°C. for 24 h. The reaction mixture was transferred to a separatory funnelwith half sat. brine (200 cm³) and extracted with ethyl acetate (3×100cm³). The combined organic layers were dried (Na₂SO₄), filtered andevaporated in vacuo to give a brown liquid. The product was put on ahigh vacuum pump to remove residual solvent. The resulting brown gum waspurified by Dry Column Vacuum Chromatography (id 4 cm; 50 cm³ fractions;0-100% EtOAc in n-heptane (v/v)—10% increments; 2-10% MeOH in EtOAc(v/v)—2% increments) to give nucleoside 10 (1.64 g, 81%) as a slightlyyellow foam.

R_(f)=0.57 (20% n-heptane in EtOAc, v/v);

ESI-MS m/z found 495.1 ([MH]⁺, calcd 495.1);

¹H NMR (CDCl₃) δ 9.02 (br s, 1H, NH), 8.07-7.99 (m, 4H, Ph), 7.62-7.58(m, 2H, Ph), 7.47-7.42 (m, 5H, Ph and H6), 5.95 (s, 1H, H1′), 5.46 (d,J=2.2 Hz, 1H, H3′), 4.93 (d, J=12.8 Hz, 1H, H5′a), 4.60 (d, J=12.8 Hz,1H, H5′b), 4.17 (d, J=2.2 Hz, 1H, H2′), 3.27 (d, J=10.6 Hz, 1H, H1″a),3.16 (d, J=10.6 Hz, 1H, H1″b), 1.55 (d, J=1.1 Hz, 3H, CH₃);

¹³C NMR (CDCl₃) δ 165.8, 165.1, 163.7 (C4, 2×PhC(O)), 150.0 (C2), 133.9,133.7, 133.6, 129.8, 129.6, 129.0, 128.8, 128.6, 128.5 (C6, 2×Ph), 110.3(C5), 91.3 (C1′), 87.5 (C4′), 72.9 (C3′), 61.3 (C5′), 50.6 (C2′), 35.6(C1″), 12.3 (CH₃);

Elemental anal. calcd for C₂₅H₂₂N₂O₇S (%): C, 60.72; H, 4.48; N, 5.66.Found: C, 60.34; H, 4.49; N, 5.35.

(1R,3R,4R,7R)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(11)

Nucleoside 10 (1.50 g, 3.0 mmol) was dissolved in methanol saturatedwith ammonia (50 cm³). The reaction flask was sealed and stirred atambient temperature for 20 h. The reaction mixture was concentrated invacuo to give a yellow gum that was purified by Dry Column VacuumChromatography (id 4 cm; 50 cm³ fractions; 0-16% MeOH in EtOAc (v/v)—1%increments) giving nucleoside 11 (0.65 g, 76%) as clear needles.

R_(f)=0.31 (10% MeOH in EtOAc, v/v);

ESI-MS m/z found 287.1 ([MH]⁺, calcd 287.1);

¹H NMR (DMSO-d₆) δ 11.32 (br s, 1H, NH), 7.96 (d, J=1.1 Hz, 1H, H6),5.95 (s, 1H, H6), 5.70 (d, J=4.2 Hz, 1H, 3′-OH), 5.62 (s, 1H, H1′), 4.49(t, J=5.3 Hz, 1H, 5′-OH), 4.20 (dd, J=4.1 and 2.1 Hz, 1H, H3′),3.77-3.67 (m, 2H, H5′), 3.42 (d, J=2.0 Hz, 1H, H2′), 2.83 (d, J=10.1 Hz,1H, H1″a), 2.64 (d, J=10.1 Hz, 1H, H1″b), 1.75 (d, J=1.1 Hz, 3H, CH₃);

¹³C NMR (DMSO-d₆) δ 163.8 (C4), 150.0 (C2), 135.3 (C6), 107.5 (C5),90.2, 89.6 (C1′ and C4′), 69.4 (C3′), 58.0 (C5′), 52.1 (C2′), 34.6(C1″), 12.4 (CH₃);

Elemental anal. calcd for C₁₁H₁₄N₂O₅S (%): C, 46.15; H, 4.93; N, 9.78.Found: C, 46.35; H, 4.91; N, 9.54.

(1R,3R,4R,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-5-methyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(12)

Nucleoside 11 (0.60 g, 2.1 mmol) was dissolved in pyridine (10 cm³).4,4′-Dimethoxytrityl chloride (0.88 g, 2.6 mmol) was added and thereaction was stirred at ambient temperature for 3 h. The reactionmixture was transferred to a separatory funnel with water (100 cm³) andextracted with ethyl acetate (100+2×50 cm³). The combined organic layerswere washed with sat. aq NaHCO₃ (100 cm³), brine (100 cm³) andevaporated to dryness in vacuo to give a viscous yellow liquid. Theproduct was redissolved in toluene (50 cm³) and concentrated in vacuo togive a yellow foam. The foam was dried on a high vacuum pump overnightand purified by Dry Column Vacuum Chromatography (id 4 cm; 50 cm³fractions; 10-100% EtOAc in n-heptane (v/v)−10% increments) givingnucleoside 12 (1.08 g, 88%) as a white foam.

R_(f)=0.24 (20% n-heptane in EtOAc, v/v);

ESI-MS m/z found 587.1 ([M-H]⁺, calcd 587.2);

¹H NMR (CDCl₃) δ 8.96 (br s, 1H, NH), 7.74 (d, J=1.1 Hz, 1H, H6),7.46-7.44 (m, 2H, Ph), 7.35-7.22 (m, 9H, Ph), 7.19-7.15 (m, 2H, Ph),6.86-6.80 (m, 2H, Ph), 5.82 (s, 1H, H1′), 4.55 (dd, J=9.3 and 2.1 Hz,1H, H3′), 3.79 (s, 6H, OCH₃), 3.71 (d, J=2.0 Hz, 1H, H2′), 3.50 (s, 2H,H5′), 2.81 (d, J=10.8 Hz, 1H, H1″a), 2.77 (d, J=10.8 Hz, 1H, H1″b), 2.69(d, J=9.2 Hz, 1H, 3′-OH), 1.42 (s, 3H, CH₃);

¹³C NMR (CDCl₃) δ 158.7 (C4), 150.1 (C2), 144.1, 135.2, 135.1, 130.1,129.1, 128.1, 128.0, 127.1, 127.0, 113.3 (C6, 3×Ph), 110.0 (C5), 90.2(C(Ph)₃), 89.6 (C1′), 87.0 (C4′), 71.7 (C3′), 60.9 (C5′), 55.2 (C2′),34.7 (C1″), 12.2 (CH₃);

Elemental anal. calcd for C₃₂H₃₂N₂O₇S.0.5H₂O (%): C, 64.31; H, 5.57; N,4.69. Found: C, 64.22; H, 5.67; N, 4.47.

(1R,3R,4R,7R)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2.2.1]heptane(13)

According to the published method (D. S. Pedersen, C. Rosenbohm, T. Koch(2002) Preparation of LNA Phosphoramidites, Synthesis, 6, 802-808)nucleoside 12 (0.78 g, 1.33 mmol) was dissolved in dichloromethane (5cm³) and a 1.0 M solution of 4,5-dicyanoimidazole in acetonitrile (0.93cm³, 0.93 mmol) was added followed by dropwise addition of2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.44 cm³, 1.33mmol). After 2 h the reaction was transferred to a separatory funnelwith dichloromethane (40 cm³) and washed with sat. aq NaHCO₃ (2×25 cm³)and brine (25 cm³). The organic layer was dried (Na₂SO₄), filtered andevaporated in vacuo to give nucleoside 13 (1.04 g, 99%) as a white foam.R_(f)=0.29 and 0.37—two diastereoisomers (20% n-heptane in EtOAc, v/v);ESI-MS m/z found 789.3 ([MH]⁺, calcd 789.3); ³¹P NMR (DMSO-d₆) δ 150.39,150.26.

Example 2 Oligonucleotide Synthesis

Oligonucleotides were synthesized using the phosphoramidite approach onan Expedite 8900/MOSS synthesizer (Multiple Oligonucleotide SynthesisSystem) at 1 or at 15 μmol. At the end of the synthesis (DMT-on) theoligonucleotides were cleaved from the solid support using aqueousammonia for 1 h at room temperature, and further deprotected for 3 h at650° C. The oligonucleotides were purified by reverse phase HPLC(RP-HPLC). After the removal of the DMT-group, the oligonucleotides werecharacterized by IE-HPLC or RP-HPLC. The identity of theoligonucleotides is confirmed by ESI-MS. See below for more details.

Preparation of the LNA Succinyl Hemiester

5′-O-Dmt-3′-hydroxy-LNA monomer (500 mg), succinic anhydride (1.2 eq.)and DMAP (1.2 eq.) were dissolved in DCM (35 mL). The reaction wasstirred at room temperature overnight. After extractions with NaH₂PO₄0.1 M pH 5.5 (2×) and brine (1×), the organic layer was further driedwith anhydrous Na₂SO₄ filtered and evaporated. The hemiester derivativewas obtained in 95% yield and was used without any further purification.

Preparation of the LNA-Support

The above prepared hemiester derivative (90 μmol) was dissolved in aminimum amount of DMF, DIEA and pyBOP (90 μmol) were added and mixedtogether for 1 min. This pre-activated mixture was combined withLCAA-CPG (500 Å, 80-120 mesh size, 300 mg) in a manual synthesizer andstirred. After 1.5 h at room temperature, the support was filtered offand washed with DMF, DCM and MeOH. After drying the loading wasdetermined to be 57 μmol/g (see Tom Brown, Dorcas J. S. Brown, “Modernmachine-aided methods of oligodeoxyribonucleotide synthesis”, in: F.Eckstein, editor. Oligonucleotides and Analogues A Practical Approach.Oxford: IRL Press, 1991: 13-14).

Elongation of the Oligonucleotide

The coupling of phosphoramidites (A(bz), G(ibu), 5-methyl-C(bz)) orT-β-cyanoethyl-phosphoramidite) is performed by using a solution of 0.1M of the 5′-O-DMT-protected amidite in acetonitrile and DCI(4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. Thethiolation is carried out by using xanthane chloride (0.01 M inacetonitrile:pyridine 10%). The rest of the reagents are the onestypically used for oligonucleotide synthesis.

Purification by RP-HPLC:

Column: XTerra, RP18, 5 μm, 7.8×50 mm column.

Eluent: Eluent A: 0.1M NH₄OAc, pH: 10.

-   -   Eluent B: Acetonitrile

Flow: 5 ml/min.

Gradient:

Time (min.) Eluent A Eluent B 0.05 min.   95% 5%  5 min. 95% 5% 12 min.65% 35%  16 min.  0% 100%  19 min.  0% 100%  21 min  100%  0%Analysis by IE-HPLC:

Column: Dionex, DNAPac PA-100, 2×250 mm column.

Eluent: Eluent A: 20 mM Tris-HCl, pH 7.6; 1 mM EDTA; 10 mM NaClO₄.

-   -   Eluent B: 20 mM Tris-HCl, pH 7.6; 1 mM EDTA; 1M NaClO₄.

Flow: 0.25 ml/min.

Gradient:

Time (min.) Eluent A Eluent B  1 min. 95% 5% 10 min. 65% 35%  11 min. 0% 100%  15 min.  0% 100%  16 min  95% 5% 21 min. 95% 5%AbbreviationsDMT: DimethoxytritylDCI: 4,5-DicyanoimidazoleDMAP: 4-DimethylaminopyridineDCM: DichloromethaneDMF: DimethylformamideTHF: TetrahydrofuraneDIEA: N,N-diisopropylethylaminePyBOP:Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphateBz: BenzoylIbu: Isobutyryl

Example 3 Test of Design of the Oligomeric Compound

It was of our interest to evaluate the antisense activity ofoligonucleotides with different designs, in order to prove theimportance of choosing the best design for an oligonucleotide targetingsurvivin. For this purpose, we set up an in vitro assay that would allowus to screen many different oligonucleotide designs by measuring theactivity of the firefly (Photinus pyralis) luciferase afterdown-regulation by antisense oligonucleotides. FIG. 1 contains anillustration of most the designs mentioned in the text. In a firstscreen, designs containing β-D-oxy-LNA, which were all targeting thesame motif within the mRNA were evaluated. Designs consisting of gapmerswith a different gap-size, a different load of phosphorothioateinternucleoside linkages, and a different load of LNA were tested.Headmers and tailmers with a different load of β-D-oxy-LNA, a differentload of phosphorothioate internucleoside linkages and a different loadof DNA were prepared. Mixmers of various compositions, which means thatbear an alternate number of units of β-D-oxy-LNA, α-L-LNA and DNA, werealso analysed in the in vitro assay. Moreover, LNA derivatives were alsoincluded in different designs, and their antisense activity wasassessed. The importance of a good design is reflected by the data thatcan be obtained in a luciferase assay. The luciferase expression levelsare measured in %, and give an indication of the antisense activity ofthe different designs containing β-D-oxy-LNA and LNA derivatives. We caneasily see that some designs are potent antisense oligonucleotides,while others give moderate to low down-regulation levels. Therefore, aclose correlation between good antisense activity and optimal design ofan oligonucleotide is very evident. We appreciated good levels ofdown-regulation with various designs. Gapmers with gaps of 7-10 nt DNAand thiolation all over the backbone or with thiolation exclusively inthe gap and PO in the flanks showed good results. These designs containβ-D-oxy-LNA or LNA derivatives. Headmers of 6 nt and 8 nt β-D-oxy-LNAalso presented good levels of down-regulation, when the phosphorothioateinternucleoside linkages are all over the backbone or only in theDNA-segment. Different mixmers gave good antisense activity in theluciferase assay. The alternate number of units of each α-L-oxy-LNA,β-D-oxy-LNA or DNA composition defines the mixmers, see FIG. 1. A mixmer3-9-3-1, which has a deoxynucleoside residue at the 3′-end showedsignificant levels of down-regulation. In a mixmer 4-1-1-5-1-1-3, weplaced two α-L-oxy-LNA residues interrupting the gap, being the flanksβ-D-oxy-LNA. Furthermore, we interrupted the gap with two α-L-oxy-LNAresidues, and substituted both flanks with α-L-oxy-LNA. Both designspresented significant levels of down-regulation. The presence ofα-L-oxy-LNA might introduce a flexible transition between theNorth-locked flanks (oxy-LNA) and the α-L-oxy-LNA residue by spiking indeoxynucleotide residues. It is also interesting to study design4-3-1-3-5 where a α-L-oxy-LNA residue interrupts the DNA stretch. Inaddition to the α-L-oxy-LNA in the gap, we also substituted two oxy-LNAresidues at the edges of the flanks with two α-L-oxy-LNA residues. Thepresence of just one β-D-oxy-LNA residue (design 4-3-1-3-5) interruptingthe stretch of DNAs in the gap results in a dramatic loss ofdown-regulation. Just by using α-L-oxy-LNA instead, the design showssignificant down-regulation at 50 nM oligonucleotide concentration. Theplacement of α-L-oxy-LNA in the junctions and one α-L-oxy-LNA in themiddle of the gap also showed down-regulation.

α-L-oxy-LNA reveals to be a potent tool enabling the construction ofdifferent mixmers, which are able to present high levels of antisenseactivity. Other mixmers such as 4-1-5-1-5 and 3-3-3-3-3-1 can also beprepared. We can easily see that some designs are potent antisenseoligonucleotides, while others give moderate to low down-regulationlevels. Therefore, again a close correlation between good antisenseactivity and optimal design of an oligonucleotide is very evident. Otherpreferred designs are (1-3-8-3-1) where DNA residues are located in theflanks with 3 β-D-oxy-LNA monomers at each side of the gap. A furtherpreferred design is (4-9-3-1) with D-oxy-LNA flanks and a 9 gap with aDNA at the 3′-end.

Assay

X1/5 Hela cell line (ECACC Ref. No: 95051229), which was stablytransfected with a “tet-off” luciferase system, was used. In the absenceof tetracycline the luciferase gene is expressed constitutively. Theexpression can be measured as light in a luminometer, when theluciferase substrate, luciferin is added. The X1/5 Hela cell line wasgrown in Minimum Essential Medium Eagle (Sigma M2279) supplemented with1× Non Essential Amino Acid (Sigma M7145), 1× Glutamax I (Invitrogen35050-038), 10% FBS calf serum, 25 μg/ml Gentamicin (Sigma G1397), 500μg/ml G418 (Invitrogen 10131-027) and 300 μg/ml Hygromycin B (Invitrogen10687-010). The X1/5 Hela cells were seeded at a density of 8000 cellsper well in a white 96 well plate (Nunc 136101) the day before thetransfection. Before the transfection, the cells were washed one timewith OptiMEM (Invitrogen) followed by addition of 40 μl OptiMEM with 2μg/ml of Lipofectamine2000 (Invitrogen). The cells were incubated for 7minutes before addition of the oligonucleotides. 10 μl ofoligonucleotide solutions were added and the cells were incubated for 4h at 37° C. and 5% CO₂. After the 4 h incubation, the cells were washedonce in OptiMEM and growth medium was added (100 μl). The luciferaseexpression was measure the next day. Luciferase expression was measuredwith the Steady-Glo luciferase assay system from Promega. 100 μl of theSteady-Glo reagent was added to each well and the plate was shaken for30 s at 700 rpm. The plate was read in Luminoskan Ascent instrument fromThermoLabsystems after 8 min of incubation to complete total lysis ofthe cells. The luciferase expression is measured as Relative Light Unitsper seconds (RLU/s). The data was processed in the Ascent software(v2.6) and graphs were drawn in SigmaPlot2001.

Example 4 In Vitro Model: Cell Culture

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. Target can be expressedendogenously or by transient or stable transfection of a nucleic acidencoding said nucleic acid.

The expression level of target nucleic acid can be routinely determinedusing, for example, Northern blot analysis, Real-Time PCR, Ribonucleaseprotection assays. The following cell types are provided forillustrative purposes, but other cell types can be routinely used,provided that the target is expressed in the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinelypassaged 2-3 times weekly.

15PC3: The human prostate cancer cell line 15PC3 was kindly donated byDr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin

A549: The human non-small cell lung cancer cell line A549 was purchasedfrom ATCC, Manassas and was cultured in DMEM (Sigma)+10% FBS+GlutamaxI+gentamicin

MCF7: The human breast cancer cell line MCF7 was purchased from ATCC andwas cultured in Eagle MEM (Sigma)+10% FBS+Glutamax I+gentamicin

SW480: The human colon cancer cell line SW480 was purchased from ATCCand was cultured in L-15 Leibovitz (Sigma)+10% FBS+Glutamax I+gentamicin

SW620: The human colon cancer cell line SW620 was purchased from ATCCand was cultured in L-15 Leibovitz (Sigma)+10% FBS+Glutamax I+gentamicin

HT29: The human prostate cancer cell line HT29 was purchased from ATCCand was cultured in McCoy's 5a MM (Sigma)+10% FBS+Glutamax I+gentamicin

NCI H23: The human non-small-cell lung cancer cell line was purchasedfrom ATCC and was cultured in RPMI 1640 with Glutamax I (Gibco)+10%FBS+HEPES+gentamicin

HCT-116: The human colon cancer cell line HCT-116 was purchased fromATCC and was cultured in McCoy's 5a MM+10% FBS+Glutamax I+gentamicin

MDA-MB-231: The human breast cancer cell line MDA-MB-231 was purchasedfrom ATCC and was cultured in L-15 Leibovitz+10% FBS+GlutamaxI+gentamicin

MDA-MB-435s: The human breast cancer cell line MDA-MB-435s was purchasedfrom ATCC and was cultured in L-15 Leibovitz+10% FBS+GlutamaxI+gentamicin

DMS273: The human small-cell lung cancer cell line DMS273 was purchasedfrom ATCC and was cultured in +10% FBS+Glutamax+gentamicin

PC3: The human prostate cancer cell line PC3 was purchased from ATCC andwas cultured in F12 Coon's with glutamine (Gibco)+10% FBS+gentamicin

U373: The human glioblastoma astrocytoma cancer cell line U373 waspurchased from ECACC and was cultured in EMEM+10%FBS+glutamax+NEAA+sodiumpyrovate+gentamicin.

HeLa Sur-GFP: Wheately, S. P. et al, Curr. Biol. 11 446-490, 2001

HUVEC-C human umbilical vein endothelial cells were purchased from ATCCand propagated according to the manufacturers instructions.

HMVEC-d (DMVEC's—dermal human microvascular endothelial cells) werepurchased from Clonetics and cultured as described by manufacturer.

HMVEC human microvascular endothelial cells were purchased fromClonetics and cultured as stated by manufacturer

Human embryonic lung fibroblasts were purchased from ATCC and culturedas described by manufacturer

HMEC-1 Human mammary epithelial cells were purchased from Clonetics andmaintained as recommended by the manufacturer.

Example 5 In Vitro Model: Treatment with Antisense Oligonucleotide

The cells were treated with oligonucleotide using the cationic liposomeformulation LipofectAMINE 2000 (Gibco) as transfection vehicle.

Cells were seeded in 12-well cell culture plates (NUNC) and treated when80-90% confluent. Oligo concentrations used ranged from 125 nM to 0.2 nMfinal concentration. Formulation of oligo-lipid complexes were carriedout essentially as described in Dean et al. (Journal of BiologicalChemistry 1994, 269, 16416-16424) using serum-free OptiMEM (Gibco) and afinal lipid concentration of 10 μg/mlLipofectAMINE 2000 in 500 μl totalvolume.

Cells were incubated at 37° C. for 4 hours and treatment was stopped byremoval of oligo-containing culture medium. Cells were washed andserum-containing media was added. After oligo treatment cells wereallowed to recover for 18 hours before they were harvested for RNA orprotein analysis.

Example 6 In Vitro Model: Extraction of RNA and cDNA Synthesis

Total RNA Isolation

Total RNA was isolated either using RNeasy mini kit (Qiagen cat. no.74104) or using the Trizol reagent (Life technologies cat. no. 15596).For RNA isolation from cell lines, RNeasy is the preferred method andfor tissue samples Trizol is the preferred method.

Total RNA was isolated from cell lines using the Qiagen RNA OPFRobot-BIO Robot 3000 according to the protocol provided by themanufacturer.

Tissue samples were homogenised using an Ultra Turrax T8 homogeniser(IKA Analysen technik) and total RNA was isolated using the Trizolreagent protocol provided by the manufacturer.

First Strand Synthesis

First strand synthesis was performed using OmniScript ReverseTranscriptase kit (cat# 205113, Qiagen) according to the manufacturersinstructions.

For each sample 0.5 μg total RNA was adjusted to 12 μl each with RNasefree H₂O and mixed with 2 μl poly (dT)₁₂₋₁₈ (SEQ ID NO: 741)(2.5 μg/ml)(Life Technologies, GibcoBRL, Roskilde, DK), 2 μl dNTP mix (5 mM eachdNTP), 2 μl 10× Buffer RT, 1 μl RNAguard™Rnase INHIBITOR (33.3 U/ml),(cat# 27-0816-01, Amersham Pharmacia Biotech, Hørsholm, DK) and 1 μlOmniScript Reverse Transcriptase (4 U/μI) followed by incubation at 37°C. for 60 minutes and heat inactivation of the enzyme at 93° C. for 5minutes.

Example 7 In Vitro Model: Analysis of Oligonucleotide Inhibition ofSurvivin Expression by Real-Time PCR

Antisense modulation of Survivin expression can be assayed in a varietyof ways known in the art. For example, Survivin mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor mRNA.

Methods of RNA isolation and RNA analysis such as Northern blot analysisis routine in the art and is taught in, for example, Current Protocolsin Molecular Biology, John Wiley and Sons.

Real-time quantitative (PCR) can be conveniently accomplished using thecommercially iQ Multi-Color Real Time PCR Detection System availablefrom BioRAD.

Real-Time Quantitative PCR Analysis of Survivin mRNA Levels

Quantitation of mRNA levels was determined by real-time quantitative PCRusing the iQ Multi-Color Real Time PCR Detection System (BioRAD)according to the manufacturers instructions.

Real-time Quantitative PCR is a technique well known in the art and istaught in for example Heid et al. Real time quantitative PCR, GenomeResearch (1996), 6: 986-994.

Platinum Quantitative PCR SuperMix UDG 2×PCR master mix was obtainedfrom Invitrogen cat# 11730. Primers and TaqMan® probes were obtainedfrom MWG-Biotech AG, Ebersberg, Germany

Probes and primers to human Survivin were designed to hybridise to ahuman Survivin sequence, using published sequence information (GenBankaccession number NM 001168, incorporated herein as SEQ ID NO:1).

For human Survivin the PCR primers were:

Assay 1

forward primer: 5′ caggtccccgctttctttg 3′ (SEQ ID NO: 727) (finalconcentration in the assay; 0.6 μM)

reverse primer: 5′ ggaggagggcgaatcaaa 3′ (SEQ ID NO: 728) (finalconcentration in the assay; 0.6 μM) and the PCR probe was: 5′FAM-ccatcatcttacgccagacttcagcc-TAMRA 3′ (SEQ ID NO: 729) (finalconcentration in the assay; 0.1 μM) Assay 2

forward primer: 5′ aaggaccaccgcatctctaca 3′ (SEQ ID NO: 730) (finalconcentration in the assay; 0.9 μM)

reverse primer: 5′ ccaagtctggctcgttctcagt 3′ (SEQ ID NO: 731) (finalconcentration in the assay; 0.6 μM) and the PCR probe was: 5′FAM-cgaggctggcttcatccactgcc-TAMRA 3′(SEQ ID NO: 732) (finalconcentration in the assay; 0.1 μM)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity was usedas an endogenous control for normalizing any variance in samplepreparation.

The sample content of human GAPDH mRNA was quantified using the humanGAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystemscat. no. 4310884E) according to the manufacturers instructions.

For quantification of mouse GAPDH mRNA the following primers and probeswere designed: Sense primer 5′aaggctgtgggcaaggtcatc 3′ (SEQ ID NO:733)(0.3 μM final concentration),

antisense primer 5′ gtcagatccacgacggacacatt (SEQ ID NO: 734) (0.6 μMfinal concentration), TaqMan probe 5′FAM-gaagctcactggcatggcatggccttccgtgttc-TAMRA 3′ (SEQ ID NO: 735) (0.2 μMfinal concentration).

Real Time PCR

The cDNA from the first strand synthesis performed as described inexample 6 was diluted 2-20 times, and analyzed by real time quantitativePCR. The primers and probe were mixed with 2× Platinum Quantitative PCRSuperMix UDG (cat. # 11730, Invitrogen) and added to 3.3 μl cDNA to afinal volume of 25 μl. Each sample was analysed in triplicates. Assaying2 fold dilutions of a cDNA that had been prepared on material purifiedfrom a cell line expressing the RNA of interest generated standardcurves for the assays. Sterile H₂O was used instead of cDNA for the notemplate control. PCR program: 50° C. for 2 minutes, 95° C. for 10minutes followed by 40 cycles of 95° C., 15 seconds, 60° C., 1 minutes.Relative quantities of target mRNA sequence were determined from thecalculated Threshold cycle using the iCycler iQ Real-time DetectionSystem software.

See FIG. 7 and Table 1, 2, 3, 4 and 5.

Example 8 In Vitro Analysis: Northern Blot Analysis of Survivin mRNALevels

Northern blot analysis was carried out by procedures well known in theart essentially as described in Current Protocols in Molecular Biology,John Wiley & Sons.

The hybridisation probe was obtained by PCR-amplification of a 373 byfragment from 1 μl cDNA obtained by reverse transcription PCR. Thereaction was carried out using primers 5′ agcacaaagccattctaagtcattg 3′(SEQ ID NO: 736) (forward) and 5′ tccatcatcttacgccagacttc 3′(SEQ ID NO:737) (reverse) at 0.5 μM final concentration each, 200 nM each dNTP, 1.5mM MgCl₂ and Platinum Taq DNA polymerase (Invitrogen cat. no.10966-018).

The DNA was amplified for 40 cycles on a Perkin Elmer 9700 thermocyclerusing the following program: 94° C. for 2 min. then 40 cycles of 94° C.for 30 sec. and 72° C. for 30 sec. with a decrease of 0.5° C. per cyclefollowed by 72° C. for 7 min.

The amplified PCR product was purified using S-400 MicroSpin columns(Amersham Pharmacia Biotech cat. no. 27-5140-01) according to themanufacturers instructions and quantified by spectrophotometry.

The hybridisation probe was labelled using Redivue™ [α-³²P]dATP 3000Ci/mmol (Amersham Pharmacia Biotech cat. # AA 0005) and Prime-It RmTlabeling kit (Stratagene cat. no. 300392) according to the manufacturersinstructions and the radioactively labeled probe was purified usingS-300 MicroSpin columns (Amersham Pharmacia Biotech cat. no.27-5130-01).

Before use, the probe was denatured at 96° C. and immediately put onice.

Samples of 2 μg of total RNA purified as described in example 6 weredenatured and size separated on a 2.2 M formaldehyde/MOPS agarose gel.RNA was transferred to positively charged nylon membrane by downwardcapillary transfer using the TurboBlotter (Schleicher & Schuell) and theRNA was immobilised to the membrane by UV crosslinking using aStratagene crosslinker. The membrane was prehybridised in ExpressHybHybridization Solution (Clontech cat. No. 8015-1) at 60° C. and theprobe was subsequently added for hybridisation. Hybridisation wascarried out at 60° C. and the blot was washed with low stringency washbuffer (2×SSC, 0.1% SDS) at room temperature and with high stringencywash buffer (0.1×SSC, 0.1% SDS) at 50° C.

The blot was exposed to Kodak storage phosphor screens and scanned in aBioRAD FX molecular imager. Survivin mRNA levels were quantified byQuantity One software (BioRAD)

Equality of RNA sample loading was assessed by stripping the blot in0.5% SDS in H₂O at 85° C. and reprobing with a labelled GAPDH(glyceraldehyde-3-phosphate dehydrogenase) probe obtained essentially asdescribed above using the primers 5′ aacggatttggtcgtatt 3′ (SEQ ID NO:739) (forward) and 5′ taagcagttggtggtgca 3′ (SEQ ID NO: 740) (reverse).See FIGS. 2 and 3. Intensity was monitored with phosphoimager Biorad,FX-scanner (see below). The tested oligomeric compounds are presented inExample 10.

Percentage down regulation of mRNA estimated from Survivin Northernblotting (data is normalised to GAPDH). Compound Seq ID 0.2 nM 1 nM 5 nM25 nM 2A 31% 34% 55% 77% 6A 22% 48% 71% 91% 9A 21% 44% 67% 64% 15A  45%79% 93% 95%

Example 9 In Vitro Analysis: Western Blot Analysis of Survivin ProteinLevels

Protein levels of Survivin can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA, RIA (Radio Immuno Assay) orfluorescence-activated cell sorting (FACS). Antibodies directed toSurvivin can be identified and obtained from a variety of sources, suchas Upstate Biotechnologies (Lake Placid, USA), Novus Biologicals(Littleton, Colo.), Santa Cruz Biotechnology (Santa Cruz, Calif.) or canbe prepared via conventional antibody generation methods.

Western Blotting:

The in vitro effect of survivin oligoes on Survivin protein levels intransfected cells was determined by Western Blotting.

Cells were transfected as described in example 5. Approximately 24 hoursafter transfection, cells were harvested, lysed in 2.5% SDS, 5 mM DTTand 6 M urea supplemented with protease inhibitor cocktail tablets(Roche). Total protein concentrations were measured using a Bradfordreagent. 150 μg total proteins was loaded onto a 12% Bis-Tris gel, runwith MOPS buffer and blotted onto a PVDF membrane according tomanufacture's recommendations (Invitrogen). After overnight incubationin blocking buffer (Invitrogen) the membrane was incubated two hourswith rabbit anti-Survivin antibodies (AF886 from R&D or Novus 500-201from Abcam) followed by one hour incubation in secondary antibodies. Achromogenic immunodetection kit (Invitrogen) was used to visualizeSurvivin. Alternatively, the membrane was incubated with HRP conjugatedrabbit immunoglobulins (DAKO) followed by incubation with ECL⁺ Plusreagent (Amersham) and visualized using VersaDoc chemiluminescensdetection system. (see FIG. 13 The tested oligomeric compounds arepresented in Example 10.)

Example 10 In Vitro Analysis: Antisense Inhibition of Human SurvivinExpression by Oligomeric Compound

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human Survivin RNA,using published sequences (GenBank accession number NM_(—)001168,incorporated herein as SEQ ID NO: 1). The oligonucleotides 16nucleotides in length are shown in Table 1 and 2. “Target site”indicates the first nucleotide number on the particular target sequenceto which the oligonucleotide binds. The preferred compounds are the LNAcontaining compounds. Table 3 shows low IC50 of four compounds.

TABLE 1 Oligomeric compounds of the invention Oligomeric compounds wereevaluated for their potential to knockdown Survivin mRNA in 15PC3 cells.The data are presented as percentage downregulation relative to mocktransfected cells. Transcript steady state was monitored by Real-timePCR and normalised to the GAPDH transcript steady state. Note that allLNA C are 5′- Methyl Cytosine Specific design of Oligomeric compound %Oligomeric Capital letters β-D-oxy-LNA % Inhibi- SEQ compound S= phosphorthioate Inhibi- tion Target ID Sequence O = —O—P(O)₂—O— tionat 5 nM site NO 5′-3′ Seq ID Small letters DNA sugar at 25 nM oligo 1722 GCAGTGGATGAAGCCA 147G_(s)C_(s)A_(s)G_(s)t_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a_(s)a_(s)G_(s)C_(s)C_(s)A85 44 148G_(s)C_(s)A_(s)G_(s)t_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a_(s)a_(s)G_(s)C_(s)C_(s)a91 149G_(O)C_(O)A_(O)G_(O)t_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a_(s)a_(s)G_(O)C_(O)C_(O)A150g_(s)c_(s)a_(s)g_(s)tg_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a_(s)a_(s)g_(s)c_(s)c_(s)a198 3 GCCAAGTCTGGCTCGT 151G_(s)C_(s)C_(s)A_(s)a_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)T_(s)C_(s)G_(s)T49 152G_(s)C_(s)C_(s)A_(s)a_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)T_(s)C_(s)G_(s)t153G_(O)C_(O)C_(O)A_(O)a_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)T_(O)C_(O)G_(O)T154g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t206 4 AACACTGGGCCAAGTC 155A_(s)A_(s)C_(s)A_(s)c_(s)t_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)A_(s)G_(s)T_(s)C74 156A_(s)A_(s)C_(s)A_(s)c_(s)t_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)A_(s)G_(s)T_(s)c91 157A_(O)A_(O)C_(O)A_(O)c_(s)t_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)A_(O)G_(O)T_(O)C158a_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)t_(s)c214 5 GCAGAAGAAACAGTGG 159G_(s)C_(s)A_(s)G_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)C_(s)T_(s)G_(s)G67 160G_(s)C_(s)A_(s)G_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)C_(s)T_(s)G_(s)g161G_(O)C_(O)A_(O)G_(O)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)C_(O)T_(O)G_(O)G162g_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g216 6 AAGCAGAAGAAACACT 163A_(s)A_(s)G_(s)C_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)C_(s)A_(s)C_(s)T88 63 164A_(s)A_(s)G_(s)C_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)C_(s)A_(s)C_(s)t79 165A_(O)A_(O)G_(O)C_(O)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)C_(O)A_(O)C_(O)T166a_(s)a_(s)g_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)c_(s)t_(s)238 7 CTCCCAGCCTTCCAGC 167C_(s)T_(s)C_(s)C_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)C_(s)A_(s)G_(s)C26 168C_(s)T_(s)C_(s)C_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)C_(s)A_(s)G_(s)c169C_(O)T_(O)C_(O)C_(O)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)C_(O)A_(O)G_(O)C170c_(s)t_(s)c_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)c_(s)a_(s)g_(s)c403 8 TTCTTTCTTCTTATTG 171T_(s)T_(s)C_(s)T_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)A_(s)T_(s)T_(s)G62 172T_(s)T_(s)C_(s)T_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)A_(s)T_(s)T_(s)g173T_(O)T_(O)C_(O)T_(O)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)A_(O)T_(O)T_(O)G174t_(s)t_(s)c_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)a_(s)t_(s)t_(s)g491 9 TGGGACCAGGCAGCTC 175T_(s)G_(s)G_(s)G_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)G_(s)C_(s)T_(s)C78 50 176T_(s)G_(s)G_(s)G_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)G_(s)C_(s)T_(s)c177T_(O)G_(O)G_(O)G_(O)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)G_(O)C_(O)T_(O)C178t_(s)t_(s)c_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)a_(s)t_(s)t_(s)g505 10 TGGTGCAGCCACTCTG 179T_(s)G_(s)G_(s)T_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)c_(s)T_(s)C_(s)T_(s)g56 180T_(s)G_(s)G_(s)T_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)c_(s)T_(O)C_(O)T_(O)G181 T_(O)G_(O)G_(O)T_(O)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)c_(s)

182t_(s)g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)c_(s)t_(s)c_(s)t_(s)g521 11 GAATAAACCCTGGAAG 183G_(s)A_(s)A_(s)T_(s)a_(s)a_(s)a_(s)c_(s)c_(s)c_(s)t_(s)g_(s)G_(s)A_(s)A_(s)G58 184G_(s)A_(s)A_(s)T_(s)a_(s)a_(s)a_(s)c_(s)c_(s)c_(s)t_(s)g_(s)G_(s)A_(s)A_(s)g185G_(O)A_(O)A_(O)T_(O)a_(s)a_(s)a_(s)c_(s)c_(s)c_(s)t_(s)g_(s)G_(O)A_(O)A_(O)G186g_(s)a_(s)a_(s)t_(s)a_(s)a_(s)a_(s)c_(s)c_(s)c_(s)t_(s)g_(s)g_(s)a_(s)a_(s)g531 12 TGGCACCAGGGAATAA 187T_(s)G_(s)G_(s)C_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)A_(s)T_(s)A_(s)A44 188T_(s)G_(s)G_(s)C_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)A_(s)T_(s)A_(s)a189T_(O)G_(O)G_(O)C_(O)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)A_(O)T_(O)A_(O)A190t_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)a_(s)t_(s)a_(s)a566 13 CTAAGACATTGCTAAG 191C_(s)T_(s)A_(s)A_(s)g_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s)c_(s)T_(s)A_(s)A_(s)G78 192C_(s)T_(s)A_(s)A_(s)g_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s)c_(s)T_(s)A_(s)A_(s)g193C_(O)T_(O)A_(O)A_(O)g_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s)c_(s)T_(O)A_(O)A_(O)G194c_(s)t_(s)a_(s)a_(s)g_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s)c_(s)t_(s)a_(s)a_(s)g579 14 TTGATCTCCTTTCCTA 195T_(s)T_(s)G_(s)A_(s)t_(s)c_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)C_(s)C_(s)T_(s)A73 196T_(s)T_(s)G_(s)A_(s)t_(s)c_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)C_(s)C_(s)T_(s)a197T_(O)T_(O)G_(O)A_(O)t_(s)c_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)C_(O)C_(O)T_(O)A198t_(s)t_(s)g_(s)a_(s)t_(s)c_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)a_(s)608 15 GCACAGTTGAAACATC 199G_(s)C_(s)A_(s)C_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)C_(s)A_(s)T_(s)C96 93 200G_(s)C_(s)A_(s)C_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)C_(s)A_(s)T_(s)c89 79 201G_(O)C_(O)A_(O)C_(O)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)C_(O)A_(O)T_(O)C202g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)t_(s)c203G_(s)C_(s)A_(s)c_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)C_(s)A_(s)T_(s)c83 78 1 16 GATTCAAATCTGGCGG 204G_(s)A_(s)T_(s)T_(s)c_(s)a_(s)a_(s)a_(s)t_(s)c_(s)t_(s)g_(s)G_(s)C_(s)G_(s)G205G_(s)A_(s)T_(s)T_(s)c_(s)a_(s)a_(s)a_(s)t_(s)c_(s)t_(s)g_(s)G_(s)C_(s)G_(s)g206G_(O)A_(O)T_(O)T_(O)c_(s)a_(s)a_(s)a_(s)t_(s)c_(s)t_(s)g_(s)G_(O)C_(O)G_(O)G207g_(s)a_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)g_(s)g17 17 TGCCAACGGGTCCCGC 208T_(s)G_(s)C_(s)C_(s)a_(s)a_(s)c_(s)g_(s)g_(s)g_(s)t_(s)c_(s)C_(s)C_(s)G_(s)C209T_(s)G_(s)C_(s)C_(s)a_(s)a_(s)c_(s)g_(s)g_(s)g_(s)t_(s)c_(s)C_(s)C_(s)G_(s)c210T_(O)G_(O)C_(O)C_(O)a_(s)a_(s)c_(s)g_(s)g_(s)g_(s)t_(s)c_(s)C_(O)C_(O)G_(O)C211t_(s)g_(s)c_(s)c_(s)a_(s)a_(s)c_(s)g_(s)g_(s)g_(s)t_(s)c_(s)c_(s)c_(s)g_(s)c33 18 CCGCCGCCGCCACCTC 212C_(s)C_(s)G_(s)C_(s)c_(s)g_(s)c_(s)c_(s)g_(s)c_(s)c_(s)a_(s)C_(s)C_(s)T_(s)C213C_(s)C_(s)G_(s)C_(s)c_(s)g_(s)c_(s)c_(s)g_(s)c_(s)c_(s)a_(s)C_(s)C_(s)T_(s)c214C_(O)C_(O)G_(O)C_(O)c_(s)g_(s)c_(s)c_(s)g_(s)c_(s)c_(s)a_(s)C_(O)C_(O)T_(O)C215c_(s)c_(s)g_(s)c_(s)c_(s)g_(s)c_(s)c_(s)g_(s)c_(s)c_(s)a_(s)c_(s)c_(s)t_(s)c49 19 CGTCGGGGCACCCATG 216C_(s)G_(s)T_(s)C_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)C_(s)A_(s)T_(s)G217C_(s)G_(s)T_(s)C_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)C_(s)A_(s)T_(s)g218C_(O)G_(O)T_(O)C_(O)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)C_(O)A_(O)T_(O)G219c_(s)g_(s)t_(s)c_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)t_(s)g65 20 GCCAGGCAGGGGGCAA 220G_(s)C_(s)C_(s)A_(s)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)g_(s)G_(s)C_(s)A_(s)A221G_(s)C_(s)C_(s)A_(s)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)g_(s)G_(s)C_(s)A_(s)a222G_(O)C_(O)C_(O)A_(O)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)g_(s)G_(O)C_(O)A_(O)A223g_(s)c_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)a81 21 TCCTTGAGAAAGGGCT 224T_(s)C_(s)C_(s)T_(s)t_(s)g_(s)a_(s)g_(s)a_(s)a_(s)a_(s)g_(s)G_(s)G_(s)C_(s)T225T_(s)C_(s)C_(s)T_(s)t_(s)g_(s)a_(s)g_(s)a_(s)a_(s)a_(s)g_(s)G_(s)G_(s)C_(s)t226T_(O)C_(O)C_(O)T_(O)t_(s)g_(s)a_(s)g_(s)a_(s)a_(s)a_(s)g_(s)G_(O)G_(O)C_(O)T227t_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)g_(s)a_(s)a_(s)a_(s)g_(s)g_(s)g_(s)c_(s)t97 22 TGTAGAGATGCGGTGG 228T_(s)G_(s)T_(s)A_(s)g_(s)a_(s)g_(s)a_(s)t_(s)g_(s)c_(s)g_(s)G_(s)T_(s)G_(s)G229T_(s)G_(s)T_(s)A_(s)g_(s)a_(s)g_(s)a_(s)t_(s)g_(s)c_(s)g_(s)G_(s)T_(s)G_(s)g230T_(O)G_(O)T_(O)A_(O)g_(s)a_(s)g_(s)a_(s)t_(s)g_(s)c_(s)g_(s)G_(O)T_(O)G_(O)G231t_(s)g_(s)t_(s)a_(s)g_(s)a_(s)g_(s)a_(s)t_(s)g_(s)c_(s)g_(s)g_(s)t_(s)g_(s)g113 23 AGGGCCAGTTCTTGAA 232A_(s)G_(s)G_(s)G_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)c_(s)t_(s)T_(s)G_(s)A_(s)A233A_(s)G_(s)G_(s)G_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)c_(s)t_(s)T_(s)G_(s)A_(s)a234A_(O)G_(O)G_(O)G_(O)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)c_(s)t_(s)T_(O)G_(O)A_(O)a235a_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)c_(s)t_(s)t_(s)g_(s)a_(s)a129 24 GCGCAGCCCTCCAAGA 236G_(s)C_(s)G_(s)C_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)c_(s)c_(s)A_(s)A_(s)G_(s)A237G_(s)C_(s)G_(s)C_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)c_(s)c_(s)A_(s)A_(s)G_(s)a238G_(O)C_(O)G_(O)C_(O)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)c_(s)c_(s)A_(O)A_(O)G_(O)A239g_(s)c_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)c_(s)c_(s)a_(s)a_(s)g_(s)a145 25 CCGCTCCGGGGTGCAG 240C_(s)C_(s)G_(s)C_(s)t_(s)c_(s)c_(s)g_(s)g_(s)g_(s)g_(s)t_(s)G_(s)C_(s)A_(s)G241C_(s)C_(s)G_(s)C_(s)t_(s)c_(s)c_(s)g_(s)g_(s)g_(s)g_(s)t_(s)G_(s)C_(s)A_(s)g242C_(O)C_(O)G_(O)C_(O)t_(s)c_(s)c_(s)g_(s)g_(s)g_(s)g_(s)t_(s)G_(O)C_(O)A_(O)G243c_(s)c_(s)g_(s)c_(s)t_(s)c_(s)c_(s)g_(s)g_(s)g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g161 26 AGCCAGCCTCGGCCAT 244A_(s)G_(s)C_(s)C_(s)a_(s)g_(s)c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)C_(s)C_(s)A_(s)T245A_(s)G_(s)C_(s)C_(s)a_(s)g_(s)c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)C_(s)C_(s)A_(s)t246A_(O)G_(O)C_(O)C_(O)a_(s)g_(s)c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)C_(O)C_(O)A_(O)T247a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)t177 27 GTGGGGCAGTGGATGA 248G_(s)T_(s)G_(s)G_(s)g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)A_(s)T_(s)G_(s)A249G_(s)T_(s)G_(s)G_(s)g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)A_(s)T_(s)G_(s)a250G_(O)T_(O)G_(O)G_(O)g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)A_(O)T_(O)G_(O)A251g_(s)t_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a193 28 GTCTGGCTCGTTCTCA 252G_(s)T_(s)C_(s)T_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)C_(s)T_(s)C_(s)A253G_(s)T_(s)C_(s)T_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)C_(s)T_(s)C_(s)a254G_(O)T_(O)C_(O)T_(O)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)C_(O)T_(O)C_(O)A255g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c_(s)a209 29 AGAAACACTGGGCCAA 256A_(s)G_(s)A_(s)A_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s)g_(s)C_(s)C_(s)A_(s)A257A_(s)G_(s)A_(s)A_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s)g_(s)C_(s)C_(s)A_(s)a258A_(O)G_(O)A_(O)A_(O)a_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s)g_(s)C_(O)C_(O)A_(O)A259a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)a225 30 AGCTCCTTGAAGCAGA 260A_(s)G_(s)C_(s)T_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)a_(s)g_(s)C_(s)A_(s)G_(s)A261A_(s)G_(s)C_(s)T_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)a_(s)g_(s)C_(s)A_(s)G_(s)a262A_(O)G_(O)C_(O)T_(O)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)a_(s)g_(s)C_(O)A_(O)G_(O)A263a_(s)g_(s)c_(s)t_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)g_(s)a241 31 TGGCTCCCAGCCTTCC 264T_(s)G_(s)G_(s)C_(s)t_(s)c_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)T_(s)T_(s)C_(s)C265T_(s)G_(s)G_(s)C_(s)t_(s)c_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)T_(s)T_(s)C_(s)c266T_(O)G_(O)G_(O)C_(O)t_(s)c_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)T_(O)T_(O)C_(O)C267t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)c257 32 CTATGGGGTCGTCATC 268C_(s)T_(s)A_(s)T_(s)g_(s)g_(s)g_(s)g_(s)t_(s)c_(s)g_(s)t_(s)C_(s)A_(s)T_(s)C269C_(s)T_(s)A_(s)T_(s)g_(s)g_(s)g_(s)g_(s)t_(s)c_(s)g_(s)t_(s)C_(s)A_(s)T_(s)c270C_(O)T_(O)A_(O)T_(O)g_(s)g_(s)g_(s)g_(s)t_(s)c_(s)g_(s)t_(s)C_(O)A_(O)T_(O)C271c_(s)t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)t_(s)c_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c273 33 TGCTTTTTATGTTCCT 272T_(s)G_(s)C_(s)T_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)t_(s)T_(s)C_(s)C_(s)T273T_(s)G_(s)C_(s)T_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)t_(s)T_(s)C_(s)C_(s)t274T_(O)G_(O)C_(O)T_(O)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)t_(s)T_(O)C_(O)C_(O)T275t_(s)g_(s)c_(s)t_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)t_(s)t_(s)c_(s)c_(s)t289 34 AGCGCAACCGGACGAA 276A_(s)G_(s)C_(s)G_(s)c_(s)a_(s)a_(s)c_(s)c_(s)g_(s)g_(s)a_(s)C_(s)G_(s)A_(s)A277A_(s)G_(s)C_(s)G_(s)c_(s)a_(s)a_(s)c_(s)c_(s)g_(s)g_(s)a_(s)C_(s)G_(s)A_(s)a278A_(O)G_(O)C_(O)G_(O)c_(s)a_(s)a_(s)c_(s)c_(s)g_(s)g_(s)a_(s)C_(O)G_(O)A_(O)A279a_(s)g_(s)c_(s)g_(s)c_(s)a_(s)a_(s)c_(s)c_(s)g_(s)g_(s)a_(s)c_(s)g_(s)a_(s)a305 35 TCTTGACAGAAAGGAA 280T_(s)C_(s)T_(s)T_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)a_(s)a_(s)G_(s)G_(s)A_(s)A281T_(s)C_(s)T_(s)T_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)a_(s)a_(s)G_(s)G_(s)A_(s)a282T_(O)C_(O)T_(O)T_(O)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)a_(s)a_(s)G_(O)G_(O)A_(O)A283t_(s)c_(s)t_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)a_(s)a_(s)g_(s)g_(s)a_(s)a321 36 AATTCTTCAAACTGCT 284A_(s)A_(s)T_(s)T_(s)c_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)c_(s)T_(s)G_(s)C_(s)T285A_(s)A_(s)T_(s)T_(s)c_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)c_(s)T_(s)G_(s)C_(s)t286A_(O)A_(O)T_(O)T_(O)c_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)c_(s)T_(O)G_(O)C_(O)T287a_(s)a_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)c_(s)t337 37 AAATTCACCAAGGGTT 288A_(s)A_(s)A_(s)T_(s)t_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)G_(s)G_(s)G_(s)T_(s)T289A_(s)A_(s)A_(s)T_(s)t_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)G_(s)G_(s)G_(s)T_(s)t290A_(O)A_(O)A_(O)T_(O)t_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)g_(s)G_(O)G_(O)T_(O)T291a_(s)a_(s)a_(s)t_(s)t_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)g_(s)g_(s)g_(s)t_(s)t353 38 CTCTGTCCAGTTTCAA 292C_(s)T_(s)C_(s)T_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)T_(s)C_(s)A_(s)A293C_(s)T_(s)C_(s)T_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)T_(s)C_(s)A_(s)a294C_(O)T_(O)C_(O)T_(O)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)T_(O)C_(O)A_(O)A295c_(s)t_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)a369 39 TTGTTCTTGGCTCTTT 296T_(s)T_(s)G_(s)T_(s)t_(s)c_(s)t_(s)t_(s)g_(s)g_(s)c_(s)t_(s)C_(s)T_(s)T_(s)T297T_(s)T_(s)G_(s)T_(s)t_(s)c_(s)t_(s)t_(s)g_(s)g_(s)c_(s)t_(s)C_(s)T_(s)T_(s)t298T_(O)T_(O)G_(O)T_(O)t_(s)c_(s)t_(s)t_(s)g_(s)g_(s)c_(s)t_(s)C_(O)T_(O)T_(O)T299t_(s)t_(s)g_(s)t_(s)t_(s)c_(s)t_(s)t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)t_(s)t_(s)t385 40 GGTTTCCTTTGCAATT 300G_(s)G_(s)T_(s)T_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)A_(s)A_(s)T_(s)T301G_(s)G_(s)T_(s)T_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)A_(s)A_(s)T_(s)t302G_(O)G_(O)T_(O)T_(O)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)A_(O)A_(O)T_(O)T303g_(s)g_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)a_(s)a_(s)t_(s)t401 41 CTTTGTTCTTATTGTT 304C_(s)T_(s)T_(s)T_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)a_(s)t_(s)T_(s)G_(s)T_(s)T305C_(s)T_(s)T_(s)T_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)a_(s)t_(s)T_(s)G_(s)T_(s)t306C_(O)T_(O)T_(O)T_(O)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)a_(s)t_(s)T_(O)G_(O)T_(O)T307c_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)a_(s)t_(s)t_(s)g_(s)t_(s)t417 42 GCAGTTTCCTCAAAIT 308G_(s)C_(s)A_(s)G_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)a_(s)A_(s)A_(s)T_(s)T309G_(s)C_(s)A_(s)G_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)a_(s)A_(s)A_(s)T_(s)t310G_(O)C_(O)A_(O)G_(O)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)a_(s)A_(O)A_(O)T_(O)T311g_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)a_(s)t_(s)t433 43 ACGGCGCACTTTCTTC 312A_(s)C_(s)G_(s)G_(s)c_(s)g_(s)c_(s)a_(s)c_(s)t_(s)t_(s)t_(s)C_(s)T_(s)T_(s)C313A_(s)C_(s)G_(s)G_(s)c_(s)g_(s)c_(s)a_(s)c_(s)t_(s)t_(s)t_(s)C_(s)T_(s)T_(s)t314A_(O)C_(O)G_(O)G_(O)c_(s)g_(s)c_(s)a_(s)c_(s)t_(s)t_(s)t_(s)C_(O)T_(O)T_(O)C315a_(s)c_(s)g_(s)g_(s)c_(s)g_(s)c_(s)a_(s)c_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c449 44 CCAGCTGCTCGATGGC 316C_(s)C_(s)A_(s)G_(s)c_(s)t_(s)g_(s)c_(s)t_(s)c_(s)g_(s)a_(s)T_(s)G_(s)G_(s)C317C_(s)C_(s)A_(s)G_(s)c_(s)t_(s)g_(s)c_(s)t_(s)c_(s)g_(s)a_(s)T_(s)G_(s)G_(s)t318C_(O)C_(O)A_(O)G_(O)c_(s)t_(s)g_(s)c_(s)t_(s)c_(s)g_(s)a_(s)T_(O)G_(O)G_(O)C319c_(s)c_(s)a_(s)g_(s)c_(s)t_(s)g_(s)c_(s)t_(s)c_(s)g_(s)a_(s)t_(s)g_(s)g_(s)c465 45 CCTCAATCCATGGCAG 320C_(s)C_(s)T_(s)C_(s)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)G_(s)C_(s)A_(s)G321C_(s)C_(s)T_(s)C_(s)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)G_(s)C_(s)A_(s)g322C_(O)C_(O)T_(O)C_(O)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)G_(O)C_(O)A_(O)g323c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)g_(s)c_(s)a_(s)g481 46 CAGCTCCGGCCAGAGG 324C_(s)A_(s)G_(s)C_(s)t_(s)c_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)G_(s)A_(s)G_(s)G325C_(s)A_(s)G_(s)C_(s)t_(s)c_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)G_(s)A_(s)G_(s)g326C_(O)A_(O)G_(O)C_(O)c_(s)c_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)G_(O)A_(O)G_(O)G327c_(s)a_(s)g_(s)c_(s)t_(s)c_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)g_(s)a_(s)g_(s)g497 47 CCACTCTGGGACCAGG 328C_(s)C_(s)A_(s)C_(s)t_(s)c_(s)t_(s)g_(s)g_(s)g_(s)a_(s)c_(s)C_(s)A_(s)G_(s)G329C_(s)C_(s)A_(s)C_(s)t_(s)c_(s)t_(s)g_(s)g_(s)g_(s)a_(s)c_(s)C_(s)A_(s)G_(s)g330C_(O)C_(O)A_(O)C_(O)t_(s)c_(s)t_(s)g_(s)g_(s)g_(s)a_(s)c_(s)C_(O)A_(O)G_(O)G331c_(s)c_(s)a_(s)c_(s)t_(s)c_(s)t_(s)g_(s)g_(s)g_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g513 48 CCTGGAAGTGGTGCAG 332C_(s)C_(s)T_(s)G_(s)g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)g_(s)t_(s)G_(s)C_(s)A_(s)G333C_(s)C_(s)T_(s)G_(s)g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)g_(s)t_(s)G_(s)C_(s)A_(s)g334C_(O)C_(O)T_(O)G_(O)g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)g_(s)t_(s)G_(O)C_(O)A_(O)G335c_(s)c_(s)t_(s)g_(s)g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g529 49 GCACCAGGGAATAAAC 336G_(s)C_(s)A_(s)C_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)a_(s)t_(s)A_(s)A_(s)A_(s)C337G_(s)C_(s)A_(s)C_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)a_(s)t_(s)A_(s)A_(s)A_(s)c338

c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)a_(s)t_(s)

339g_(s)c_(s)a_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)a_(s)t_(s)a_(s)a_(s)a_(s)c545 50 CACAGGAAGGCTGGTG 340

g_(s)g_(s)a_(s)a_(s)g_(s)g_(s)c_(s)t_(s)

341

g_(s)g_(s)a_(s)a_(s)g_(s)g_(s)c_(s)t_(s)

342

g_(s)g_(s)a_(s)a_(s)g_(s)g_(s)c_(s)t_(s)

343c_(s)a_(s)c_(s)a_(s)g_(s)g_(s)a_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)g_(s)t_(s)g561 51 ACATTGCTAAGGGGCC 344

t_(s)g_(s)c_(s)t_(s)a_(s)a_(s)g_(s)g_(s)

345

t_(s)g_(s)c_(s)t_(s)a_(s)a_(s)g_(s)g_(s)

346

t_(s)g_(s)c_(s)t_(s)a_(s)a_(s)g_(s)g_(s)

347a_(s)c_(s)a_(s)t_(s)t_(s)g_(s)c_(s)t_(s)a_(s)a_(s)g_(s)g_(s)g_(s)g_(s)c_(s)c577 52 GATCTCCTTTCCTAAG 348

t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s)c_(s)

349

t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s)c_(s)

350

t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s)c_(s)

351g_(s)a_(s)t_(s)c_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)a_(s)a_(s)g593 53 CTAATTTGAAAATGTT 352

t_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)a_(s)

353

t_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)a_(s)

354

t_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)a_(s)

355c_(s)t_(s)a_(s)a_(s)t_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)a_(s)t_(s)g_(s)t_(s)t609 54 AGCACAGTTGAAACAT 356

c_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)

357

c_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)

358

c_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)

359a_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)t625 55 TTCAAGACAAAACAGG 360

a_(s)g_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)

361

a_(s)g_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)

362

a_(s)g_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)

363t_(s)t_(s)c_(s)a_(s)a_(s)g_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)c_(s)a_(s)g_(s)g641 56 CACCTCTGGTGCCACT 364

t_(s)c_(s)t_(s)g_(s)g_(s)t_(s)g_(s)c_(s)

365

t_(s)c_(s)t_(s)g_(s)g_(s)t_(s)g_(s)c_(s)

366

t_(s)c_(s)t_(s)g_(s)g_(s)t_(s)g_(s)c_(s)

367c_(s)a_(s)c_(s)c_(s)t_(s)c_(s)t_(s)g_(s)g_(s)t_(s)g_(s)c_(s)c_(s)a_(s)c_(s)t657 57 GCTGCACAGGCAGAAG 368

c_(s)a_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)

369

c_(s)a_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)

370

c_(s)a_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)

371g_(s)c_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g673 58 GTTACCAGCAGCACCC 372

c_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)

373

c_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)

374

c_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)

375g_(s)t_(s)t_(s)a_(s)c_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)a_(s)c_(s)c_(s)c689 59 GAGAGAAGCAGCCACT 376

g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)

377

g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)

378

g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)

379g_(s)a_(s)g_(s)a_(s)g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)c_(s)t705 60 AAAAAAGAGAGAGAGA 380

a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

381

a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

382

a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

383a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a721 61 GCAAAAATGAGCCCCC 384

a_(s)a_(s)a_(s)t_(s)g_(s)a_(s)g_(s)c_(s)

385

a_(s)a_(s)a_(s)t_(s)g_(s)a_(s)g_(s)c_(s)

386

a_(s)a_(s)a_(s)t_(s)g_(s)a_(s)g_(s)c_(s)

387g_(s)c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)t_(s)g_(s)a_(s)g_(s)c_(s)c_(s)c_(s)c_(s)c737 62 CCCGGGAATCAAAACA 388

g_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)a_(s)

389

g_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)a_(s)

390

g_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)a_(s)

391c_(s)c_(s)c_(s)g_(s)g_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)a_(s)a_(s)a_(s)c_(s)a753 63 CTTCTCACCTGGTAAG 392

t_(s)c_(s)a_(s)c_(s)c_(s)t_(s)g_(s)g_(s)

393

t_(s)c_(s)a_(s)c_(s)c_(s)t_(s)g_(s)g_(s)

394

t_(s)c_(s)a_(s)c_(s)c_(s)t_(s)g_(s)g_(s)

395c_(s)t_(s)t_(s)c_(s)t_(s)c_(s)a_(s)c_(s)c_(s)t_(s)g_(s)g_(s)t_(s)a_(s)a_(s)g769 64 CCTTCTTCCTCCCTCA 396

c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)c_(s)

397

c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)c_(s)

398

c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)c_(s)

399c_(s)c_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)c_(s)c_(s)t_(s)c_(s)a785 65 AGCAAAAGGGACAGTG 400

a_(s)a_(s)a_(s)g_(s)g_(s)g_(s)a_(s)c_(s)

401

a_(s)a_(s)a_(s)g_(s)g_(s)g_(s)a_(s)c_(s)

402

a_(s)a_(s)a_(s)g_(s)g_(s)g_(s)a_(s)c_(s)

403a_(s)g_(s)c_(s)a_(s)a_(s)a_(s)a_(s)g_(s)g_(s)g_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g801 66 CAAAGCTGTCAGCTCT 404

g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)a_(s)g_(s)

405

g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)a_(s)g_(s)

406

g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)a_(s)g_(s)

407c_(s)a_(s)a_(s)a_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)a_(s)g_(s)c_(s)t_(s)c_(s)t817 67 GCTCTGCCCACGCGAA 408

t_(s)g_(s)c_(s)c_(s)c_(s)a_(s)c_(s)

409

t_(s)g_(s)c_(s)c_(s)c_(s)a_(s)c_(s)

410

t_(s)g_(s)c_(s)c_(s)c_(s)a_(s)c_(s)

411g_(s)c_(s)t_(s)c_(s)t_(s)g_(s)c_(s)c_(s)c_(s)a_(s)c_(s)g_(s)c_(s)g_(s)a_(s)a833 68 ACATTCACTGTGGAAG 412

t_(s)c_(s)a_(s)c_(s)t_(s)g_(s)t_(s)g_(s)

413

t_(s)c_(s)a_(s)c_(s)t_(s)g_(s)t_(s)g_(s)

414

t_(s)c_(s)a_(s)c_(s)t_(s)g_(s)t_(s)g_(s)

415a_(s)ac_(s)a_(s)t_(s)t_(s)c_(s)a_(s)c_(s)t_(s)g_(s)t_(s)g_(s)g_(s)a_(s)a_(s)g849 69 AACATGAGGTCCAGAC 416

t_(s)g_(s)a_(s)g_(s)g_(s)t_(s)c_(s)c_(s)

417

t_(s)g_(s)a_(s)g_(s)g_(s)t_(s)c_(s)c_(s)

418

t_(s)g_(s)a_(s)g_(s)g_(s)t_(s)c_(s)c_(s)

419a_(s)a_(s)c_(s)a_(s)t_(s)g_(s)a_(s)g_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)a_(s)c865 70 CTGTGACAGCCTCAAC 420

g_(s)a_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)

421

g_(s)a_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)

422

g_(s)a_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)

423c_(s)t_(s)g_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)c881 71 AAGTCCACACTCAGGA 424

c_(s)c_(s)a_(s)c_(s)a_(s)c_(s)t_(s)c_(s)

425

c_(s)c_(s)a_(s)c_(s)a_(s)c_(s)t_(s)c_(s)

426

c_(s)c_(s)a_(s)c_(s)a_(s)c_(s)t_(s)c_(s)

427a_(s)a_(s)g_(s)t_(s)c_(s)c_(s)a_(s)c_(s)a_(s)c_(s)t_(s)c_(s)a_(s)g_(s)g_(s)a897 72 TCAACAGGCACCTGCC 428

c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)

429

c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)

430

c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)

431t_(s)c_(s)a_(s)a_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)t_(s)g_(s)c_(s)c913 73 AACCTGCAGCTCAGAT 432

t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)t_(s)c_(s)

433

t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)t_(s)c_(s)

434

t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)t_(s)c_(s)

435a_(s)a_(s)c_(s)c_(s)t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)t_(s)c_(s)a_(s)g_(s)a_(s)t929 74 GGTGTGACAGATAAGG 436

t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)t_(s)

437

t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)t_(s)

438

t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)t_(s)

439g_(s)g_(s)t_(s)g_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)t_(s)a_(s)a_(s)g_(s)g945 75 CCTCTGAGGAGGCACA 440

t_(s)g_(s)a_(s)g_(s)g_(s)a_(s)g_(s)g_(s)

441

t_(s)g_(s)a_(s)g_(s)g_(s)a_(s)g_(s)g_(s)

442

t_(s)g_(s)a_(s)g_(s)g_(s)a_(s)g_(s)g_(s)

443c_(s)c_(s)t_(s)c_(s)t_(s)g_(s)a_(s)g_(s)g_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a961 76 ACAACAAAAAAACTGT 444

c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)

445

c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)

446

c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)

447a_(s)c_(s)a_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)t977 77 AAAACAAAAAAACACA 448

c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)

449

c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)

450

c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)

451a_(s)a_(s)a_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)c_(s)a_(s)c_(s)a993 78 CATCTACCAAAAAAAA 452

t_(s)a_(s)c_(s)c_(s)a_(s)a_(s)a_(s)a_(s)

453

t_(s)a_(s)c_(s)c_(s)a_(s)a_(s)a_(s)a_(s)

454

t_(s)a_(s)c_(s)c_(s)a_(s)a_(s)a_(s)a_(s)

455c_(s)a_(s)t_(s)c_(s)t_(s)a_(s)c_(s)c_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a_(s)a1009 79 TCACACACAAGTCATG 456

a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)

457

a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)

458

a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)

459t_(s)c_(s)a_(s)c_(s)a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)c_(s)a_(s)t_(s)g1025 80 TGTCTCCATTCTCTCA 460

t_(s)c_(s)c_(s)a_(s)t_(s)t_(s)c_(s)t_(s)

461

t_(s)c_(s)c_(s)a_(s)t_(s)t_(s)c_(s)t_(s)

462

t_(s)c_(s)c_(s)a_(s)t_(s)t_(s)c_(s)t_(s)

463t_(s)g_(s)t_(s)c_(s)t_(s)c_(s)c_(s)a_(s)t_(s)t_(s)c_(s)t_(s)c_(s)t_(s)c_(s)a1041 81 GAGGAGCCAGGGACTC 464

a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)

465

a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)

466

a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)

467g_(s)a_(s)g_(s)g_(s)a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)c_(s)t_(s)c1057 82 ATGTTGTTAAACAGTA 468

t_(s)g_(s)t_(s)t_(s)a_(s)a_(s)a_(s)c_(s)

469

t_(s)g_(s)t_(s)t_(s)a_(s)a_(s)a_(s)c_(s)

470

t_(s)g_(s)t_(s)t_(s)a_(s)a_(s)a_(s)c_(s)

471a_(s)t_(s)g_(s)t_(s)t_(s)g_(s)t_(s)t_(s)a_(s)a_(s)a_(s)c_(s)a_(s)g_(s)t_(s)a1073 83 ACAAAATAAGAAAGCC 472

a_(s)a_(s)t_(s)a_(s)a_(s)g_(s)a_(s)a_(s)

473

a_(s)a_(s)t_(s)a_(s)a_(s)g_(s)a_(s)a_(s)

474

a_(s)a_(s)t_(s)a_(s)a_(s)g_(s)a_(s)a_(s)

475a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)t_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)g_(s)c_(s)c1089 84 TGAATTAACAATTCAA 476

t_(s)t_(s)a_(s)a_(s)c_(s)a_(s)a_(s)t_(s)

477

t_(s)t_(s)a_(s)a_(s)c_(s)a_(s)a_(s)t_(s)

478

t_(s)t_(s)a_(s)a_(s)c_(s)a_(s)a_(s)t_(s)

479t_(s)g_(s)a_(s)a_(s)t_(s)t_(s)a_(s)a_(s)c_(s)a_(s)a_(s)t_(s)t_(s)c_(s)a_(s)a1105 85 AGTTTGTGCTATTCTG 480

t_(s)g_(s)t_(s)g_(s)c_(s)t_(s)a_(s)t_(s)

481

t_(s)g_(s)t_(s)g_(s)c_(s)t_(s)a_(s)t_(s)

482

t_(s)g_(s)t_(s)g_(s)c_(s)t_(s)a_(s)t_(s)

483a_(s)g_(s)t_(s)t_(s)t_(s)g_(s)t_(s)g_(s)c_(s)t_(s)a_(s)t_(s)t_(s)c_(s)t_(s)g1121 86 GCTTAGTTTTAATTGT 484

a_(s)g_(s)t_(s)t_(s)t_(s)t_(s)a_(s)a_(s)

485

a_(s)g_(s)t_(s)t_(s)t_(s)t_(s)a_(s)a_(s)

486

a_(s)g_(s)t_(s)t_(s)t_(s)t_(s)a_(s)a_(s)

487g_(s)c_(s)t_(s)t_(s)a_(s)g_(s)t_(s)t_(s)t_(s)t_(s)a_(s)a_(s)t_(s)t_(s)g_(s)t1137 87 CTTAGAATGGCTTTGT 488

g_(s)a_(s)a_(s)t_(s)g_(s)g_(s)c_(s)t_(s)

489

g_(s)a_(s)a_(s)t_(s)g_(s)g_(s)c_(s)t_(s)

490

g_(s)a_(s)a_(s)t_(s)g_(s)g_(s)c_(s)t_(s)

491c_(s)t_(s)t_(s)a_(s)g_(s)a_(s)a_(s)t_(s)g_(s)g_(s)c_(s)t_(s)t_(s)t_(s)g_(s)t1153 88 CCCGTTTCCCCAATGA 492

t_(s)t_(s)t_(s)c_(s)c_(s)c_(s)c_(s)a_(s)

493

t_(s)t_(s)t_(s)c_(s)c_(s)c_(s)c_(s)a_(s)

494

t_(s)t_(s)t_(s)c_(s)c_(s)c_(s)c_(s)a_(s)

495c_(s)c_(s)c_(s)g_(s)t_(s)t_(s)t_(s)c_(s)c_(s)c_(s)c_(s)a_(s)a_(s)t_(s)g_(s)a1169 89 TCCACCTGAAGTTCAC 496

c_(s)c_(s)t_(s)g_(s)a_(s)a_(s)g_(s)t_(s)

497

c_(s)c_(s)t_(s)g_(s)a_(s)a_(s)g_(s)t_(s)

498

c_(s)c_(s)t_(s)g_(s)a_(s)a_(s)g_(s)t_(s)

499t_(s)c_(s)c_(s)a_(s)c_(s)c_(s)t_(s)g_(s)a_(s)a_(s)g_(s)t_(s)t_(s)c_(s)a_(s)c1185 90 CTATTCTGTCTCCTCA 500

t_(s)c_(s)t_(s)g_(s)t_(s)c_(s)t_(s)c_(s)

501

t_(s)c_(s)t_(s)g_(s)t_(s)c_(s)t_(s)c_(s)

502

t_(s)c_(s)t_(s)g_(s)t_(s)c_(s)t_(s)c_(s)

503c_(s)t_(s)a_(s)t_(s)t_(s)c_(s)t_(s)g_(s)t_(s)c_(s)t_(s)c_(s)c_(s)t_(s)c_(s)a1201 91 GACGCTTCCTATCACT 504

c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)a_(s)t_(s)

505

c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)a_(s)t_(s)

506

c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)a_(s)t_(s)

507g_(s)a_(s)c_(s)g_(s)c_(s)t_(s)t_(s)c_(s)c_(s)t_(s)a_(s)t_(s)c_(s)a_(s)c_(s)t1217 92 AAAGGAGTATCTGCCA 508

g_(s)a_(s)g_(s)t_(s)a_(s)t_(s)c_(s)t_(s)

509

g_(s)a_(s)g_(s)t_(s)a_(s)t_(s)c_(s)t_(s)

510

g_(s)a_(s)g_(s)t_(s)a_(s)t_(s)c_(s)t_(s)

511a_(s)a_(s)a_(s)g_(s)g_(s)a_(s)g_(s)t_(s)a_(s)t_(s)c_(s)t_(s)g_(s)c_(s)c_(s)a1233 93 TCACACAGCAGTGGCA 512

a_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)t_(s)

513

a_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)t_(s)

514

a_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)t_(s)

515t_(s)c_(s)a_(s)c_(s)a_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)c_(s)a1249 94 CACTGGGCCTGTCTAA 516

g_(s)g_(s)g_(s)c_(s)c_(s)t_(s)g_(s)t_(s)

517

g_(s)g_(s)g_(s)c_(s)c_(s)t_(s)g_(s)t_(s)

518

g_(s)g_(s)g_(s)c_(s)c_(s)t_(s)g_(s)t_(s)

519c_(s)a_(s)c_(s)t_(s)g_(s)g_(s)g_(s)c_(s)c_(s)t_(s)g_(s)t_(s)c_(s)t_(s)a_(s)a1265 95 CATGTGCCCCGCGGCT 520

t_(s)g_(s)c_(s)c_(s)c_(s)c_(s)g_(s)c_(s)

521

t_(s)g_(s)c_(s)c_(s)c_(s)c_(s)g_(s)c_(s)

522

t_(s)g_(s)c_(s)c_(s)c_(s)c_(s)g_(s)c_(s)

523c_(s)a_(s)t_(s)g_(s)t_(s)g_(s)c_(s)c_(s)c_(s)c_(s)g_(s)c_(s)g_(s)g_(s)c_(s)t1281 96 AGGGAGGAGCGGCCAG 524

a_(s)g_(s)g_(s)a_(s)g_(s)c_(s)g_(s)g_(s)

525

a_(s)g_(s)g_(s)a_(s)g_(s)c_(s)g_(s)g_(s)

526

a_(s)g_(s)g_(s)a_(s)g_(s)c_(s)g_(s)g_(s)

527a_(s)g_(s)g_(s)g_(s)a_(s)g_(s)g_(s)a_(s)g_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)g1297 97 CCACTGCCTTTTTCTG 528

t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)t_(s)t_(s)

529

t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)t_(s)t_(s)

530

t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)t_(s)t_(s)

531c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)t_(s)t_(s)t_(s)c_(s)t_(s)g1313 98 TTAAAAAGGATTTAGG 532

a_(s)a_(s)a_(s)g_(s)g_(s)a_(s)t_(s)t_(s)

533

a_(s)a_(s)a_(s)g_(s)g_(s)a_(s)t_(s)t_(s)

534

a_(s)a_(s)a_(s)g_(s)g_(s)a_(s)t_(s)t_(s)

535t_(s)t_(s)a_(s)a_(s)a_(s)a_(s)a_(s)g_(s)g_(s)a_(s)t_(s)t_(s)t_(s)a_(s)g_(s)g1329 99 CATCGAGCCAAGTCAT 536

g_(s)a_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)

537

g_(s)a_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)

538

g_(s)a_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)

539c_(s)a_(s)t_(s)c_(s)g_(s)a_(s)g_(s)c_(s)c_(s)a_(s)a_(s)g_(s)t_(s)c_(s)a_(s)t1345 100 AGCCAGTCCCCCACAG 540

a_(s)g_(s)t_(s)c_(s)c_(s)c_(s)c_(s)c_(s)

541

a_(s)g_(s)t_(s)c_(s)c_(s)c_(s)c_(s)c_(s)

542

a_(s)g_(s)t_(s)c_(s)c_(s)c_(s)c_(s)c_(s)

543a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)t_(s)c_(s)c_(s)c_(s)c_(s)c_(s)a_(s)c_(s)a_(s)g1361 101 CGGCCTGCAGCAGCCC 544

c_(s)t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)a_(s)

545

c_(s)t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)a_(s)

546

c_(s)t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)a_(s)

547c_(s)g_(s)g_(s)c_(s)c_(s)t_(s)g_(s)c_(s)a_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)c1377 102 TGGGCTGACAGACACA 548

c_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

549

c_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

550

c_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

551t_(s)g_(s)g_(s)g_(s)c_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)c_(s)a_(s)c_(s)a1393 103 TGACAGATGTGAAGGT 552

a_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)a_(s)

553

a_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)a_(s)

554

a_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)a_(s)

555t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s)g_(s)t1409 104 CCCCGTGTGGAGAACG 556

g_(s)t_(s)g_(s)t_(s)g_(s)g_(s)a_(s)g_(s)

557

g_(s)t_(s)g_(s)t_(s)g_(s)g_(s)a_(s)g_(s)

558

g_(s)t_(s)g_(s)t_(s)g_(s)g_(s)a_(s)g_(s)

559c_(s)c_(s)c_(s)c_(s)g_(s)t_(s)g_(s)t_(s)g_(s)g_(s)a_(s)g_(s)a_(s)a_(s)c_(s)g1425 105 GCGGACTGCGTCTCTC 560

_(s)c_(s)t_(s)g_(s)c_(s)g_(s)t_(s)c_(s)

561

a_(s)c_(s)t_(s)g_(s)c_(s)g_(s)t_(s)c_(s)

562

a_(s)c_(s)t_(s)g_(s)c_(s)g_(s)t_(s)c_(s)

563g_(s)c_(s)g_(s)g_(s)a_(s)c_(s)t_(s)g_(s)c_(s)g_(s)t_(s)c_(s)t_(s)c_(s)t_(s)c1441 106 GAAAGCGGGGACCTGG 564

g_(s)c_(s)g_(s)g_(s)g_(s)g_(s)a_(s)c_(s)

565

g_(s)c_(s)g_(s)g_(s)g_(s)g_(s)a_(s)c_(s)

566

g_(s)c_(s)g_(s)g_(s)g_(s)g_(s)a_(s)c_(s)

567g_(s)a_(s)a_(s)a_(s)g_(s)c_(s)g_(s)g_(s)g_(s)g_(s)a_(s)c_(s)c_(s)t_(s)g_(s)g1457 107 AGGTGCTGCCTCCAAA 568

g_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)c_(s)

569

g_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)c_(s)

570

g_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)c_(s)

571a_(s)g_(s)c_(s)t_(s)g_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)c_(s)c_(s)a_(s)a_(s)a1473 108 ACTTCAGCCCTGCGGG 572

c_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)g_(s)

573

c_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)g_(s)

574

c_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)g_(s)

575a_(s)c_(s)t_(s)t_(s)c_(s)a_(s)g_(s)c_(s)c_(s)c_(s)t_(s)g_(s)c_(s)g_(s)g_(s)g1489 109 CATCATCTTACGCCAG 576

a_(s)t_(s)c_(s)t_(s)t_(s)a_(s)c_(s)g_(s)

577

a_(s)t_(s)c_(s)t_(s)t_(s)a_(s)c_(s)g_(s)

578

a_(s)t_(s)c_(s)t_(s)t_(s)a_(s)c_(s)g_(s)

579c_(s)a_(s)t_(s)c_(s)a_(s)t_(s)c_(s)t_(s)t_(s)a_(s)c_(s)g_(s)c_(s)c_(s)a_(s)g1505 110 GAGGGCGAATCAAATC 580

g_(s)c_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)

581

g_(s)c_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)

582

g_(s)c_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)

583g_(s)a_(s)g_(s)g_(s)g_(s)c_(s)g_(s)a_(s)a_(s)t_(s)c_(s)a_(s)a_(s)a_(s)t_(s)c1521 111 GCTCTATGACAGGGAG 584

t_(s)a_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)

585

t_(s)a_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)

586

t_(s)a_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)

587g_(s)c_(s)t_(s)c_(s)t_(s)a_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)g_(s)g_(s)a_(s)g1537 112 AACAATCCACCCTGCA 588

a_(s)t_(s)c_(s)c_(s)a_(s)c_(s)c_(s)c_(s)

589

a_(s)t_(s)c_(s)c_(s)a_(s)c_(s)c_(s)c_(s)

590

a_(s)t_(s)c_(s)c_(s)a_(s)c_(s)c_(s)c_(s)

591a_(s)a_(s)c_(s)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)c_(s)c_(s)c_(s)t_(s)g_(s)c_(s)a1553 113 TTTCCAGGGAAGCTGT 592

c_(s)a_(s)g_(s)c_(s)g_(s)a_(s)a_(s)g_(s)

593

c_(s)a_(s)g_(s)c_(s)g_(s)a_(s)a_(s)g_(s)

594

c_(s)a_(s)g_(s)c_(s)g_(s)a_(s)a_(s)g_(s)

595t_(s)t_(s)t_(s)c_(s)c_(s)a_(s)g_(s)c_(s)g_(s)a_(s)a_(s)g_(s)c_(s)t_(s)g_(s)t1569 114 AGATGACCTCCAGAGG 596

g_(s)a_(s)c_(s)c_(s)t_(s)c_(s)c_(s)a_(s)

597

g_(s)a_(s)c_(s)c_(s)t_(s)c_(s)c_(s)a_(s)

598

g_(s)a_(s)c_(s)c_(s)t_(s)c_(s)c_(s)a_(s)

599a_(s)g_(s)a_(s)t_(s)g_(s)a_(s)c_(s)c_(s)t_(s)c_(s)c_(s)a_(s)g_(s)a_(s)g_(s)g1585 115 TTCTCAGGAACAGCCG 600

c_(s)a_(s)g_(s)g_(s)a_(s)a_(s)c_(s)a_(s)

601

c_(s)a_(s)g_(s)g_(s)a_(s)a_(s)c_(s)a_(s)

602

c_(s)a_(s)g_(s)g_(s)a_(s)a_(s)c_(s)a_(s)

603t_(s)t_(s)c_(s)t_(s)c_(s)a_(s)g_(s)g_(s)a_(s)a_(s)c_(s)a_(s)g_(s)c_(s)c_(s)g1601 116 ATGACAGGCTTTTTAT 604

c_(s)a_(s)g_(s)g_(s)c_(s)t_(s)t_(s)t_(s)

605

c_(s)a_(s)g_(s)g_(s)c_(s)t_(s)t_(s)t_(s)

606

c_(s)a_(s)g_(s)g_(s)c_(s)t_(s)t_(s)t_(s)

607a_(s)t_(s)g_(s)a_(s)c_(s)a_(s)g_(s)g_(s)c_(s)t_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t

TABLE 2 Oligomeric compounds of the invention Oligomeric compounds wereevaluated for their potential to knockdown Survivin mRNA in 15PC3 cells.The data are presented as percentage downregulation relative to mocktransfected cells. Transcript steady state was monitored by Real-timePCR and normalised to the GAPDH transcript steady state. Note that allLNA C are 5′- Methyl-Cytosine. Specific design of Oligomeric compound %Oligomeric Capital letters β-D-oxy-LNA % Inhibi- Seq compound S= phosphorthioate Inhibi- tion Target ID Sequence Seq O = —O—P(O)₂—O—tion at 5 nM site NO: 5′-3′ ID Small letters DNA sugar at 25 nM oligo 62(c) 117 AGGCAGGGGGCAACGT 608

a_(s)g_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)

<20 <20 609

a_(s)g_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)

t 610

a_(s)g_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)

611a_(s)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)a_(s)c_(s)g_(s)t119(c) 118 CCAAGAAGGGCCAGTT 612

g_(s)a_(s)a_(s)g_(s)g_(s)g_(s)c_(s)c_(s)

87 33 613

g_(s)a_(s)a_(s)g_(s)g_(s)g_(s)c_(s)c_(s)

t 614

g_(s)a_(s)a_(s)g_(s)g_(s)g_(s)c_(s)c_(s)

615c_(s)c_(s)a_(s)a_(s)g_(s)a_(s)a_(s)g_(s)g_(s)g_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t190(c) 119 TGGCTCGTTCTCACTG 616

t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c_(s)

79 27 617

t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c_(s)

g 618

t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c_(s)

619t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c_(s)a_(s)g_(s)t_(s)g193(c) 120 GTCTGGCTCGTTCTCA 620

g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)

84 47 621

g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)

a 622

g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)

623g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c_(s)a194(c) 121 AGTCTGGCTCGTTCTC 624

t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)

75 49 625

t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)

626

t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)

627a_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)c_(s)t_(s)c_(s)g_(s)t_(s)t_(s)c_(s)t_(s)c168(c) 122 TGGATGAAGCCAGCCT 628

g_(s)a_(s)a_(s)g_(s)c_(s)c_(s)a_(s)

67 41 629

t_(s)g_(s)a_(s)a_(s)g_(s)c_(s)c_(s)a_(s)

t 630

t_(s)g_(s)a_(s)a_(s)g_(s)c_(s)c_(s)a_(s)

631t_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a_(s)a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t215(c) 123 AGCAGAAGAAACACTG 632

g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)

85 26 633

g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)

g 634

g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)

635a_(s)g_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)a_(s)a_(s)c_(s)a_(s)c_(s)t_(s)g261(c) 124 TCCTCTATGGGGTCGT 636

c_(s)t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)

23 <20 637

c_(s)t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)

638

c_(s)t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)

639t_(s)c_(s)c_(s)t_(s)c_(s)t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)t_(s)c_(s)g_(s)t286(c) 125 GCAACCGGACGAATGC 640

c_(s)c_(s)g_(s)g_(s)a_(s)c_(s)g_(s)a_(s)

64 <20 641

c_(s)c_(s)g_(s)g_(s)a_(s)c_(s)g_(s)a_(s)

c 642

c_(s)c_(s)g_(s)g_(s)a_(s)c_(s)g_(s)a_(s)

643g_(s)c_(s)a_(s)a_(s)c_(s)c_(s)g_(s)g_(s)a_(s)c_(s)g_(s)a_(s)a_(s)t_(s)g_(s)c267(c) 126 TTATGTTCCTCTATGG 644

g_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)t_(s)

53 <20 645

g_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)t_(s)

g 646

g_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)t_(s)

647t_(s)t_(s)a_(s)t_(s)g_(s)t_(s)t_(s)c_(s)c_(s)t_(s)c_(s)t_(s)a_(s)t_(s)g_(s)g325(c) 127 GGTTAATTCTTCAAAC 648

a_(s)a_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)

17 <20 649

a_(s)a_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)

c 650

a_(s)a_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)

651g_(s)g_(s)t_(s)t_(s)a_(s)a_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)c353(c) 128 GTCTGTCCAGTTTCAA 652

g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)

76 60 653

g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)

a 77 654

g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)

655c_(s)t_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)a375(c) 129 GCAATTTTGTTCTTGG 656

t_(s)t_(s)t_(s)t_(s)g_(s)t_(s)t_(s)c_(s)

73 49 657

t_(s)t_(s)t_(s)t_(s)g_(s)t_(s)t_(s)c_(s)

g 658

t_(s)t_(s)t_(s)t_(s)g_(s)t_(s)t_(s)c_(s)

659g_(s)c_(s)a_(s)a_(s)t_(s)t_(s)t_(s)t_(s)g_(s)t_(s)t_(s)c_(s)t_(s)t_(s)g_(s)g464(c) 130 CTCAATCCATGGCAGC 660

a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)g_(s)

77 40 661

a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)g_(s)

c 662

a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)g_(s)

663c_(s)t_(s)c_(s)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)t_(s)g_(s)g_(s)c_(s)a_(s)g_(s)c159(c) 131 CCAGCCTCGGCCATCC 664

c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)c_(s)c_(s)

80 29 665

c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)c_(s)c_(s)

c 94 666

c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)c_(s)c_(s)

667c_(s)c_(s)a_(s)g_(s)c_(s)c_(s)t_(s)c_(s)g_(s)g_(s)c_(s)c_(s)a_(s)t_(s)c_(s)c350(c) 132 TGTCCAGTTTCAAAAA 668

c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)

<20 <20 669

c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)

a 670

c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)

671t_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)a_(s)a351(c) 133 CTGTCCAGTTTCAAAA 672

c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)

<20 <20 673

_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)

a 674

c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)

675c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)a_(s)a_(s)a_(s)a 47(c) 134 TCGGGGCACCCATGCC 676

g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)

677

g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)

678

g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)

679t_(s)c_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)t_(s)g_(s)c_(s)c456(c) 135 ATGGCAGCCAGCTGCT 680

c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)c_(s)

681

c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)c_(s)

682

c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)c_(s)

683a_(s)t_(s)g_(s)g_(s)c_(s)a_(s)g_(s)c_(s)c_(s)a_(s)g_(s)c_(s)t_(s)g_(s)c_(s)t470(c) 136 AGAGGCCTCAATCCAT 684

g_(s)c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)t_(s)

685

g_(s)c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)t_(s)

686

g_(s)c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)t_(s)

687a_(s)g_(s)a_(s)g_(s)g_(s)c_(s)c_(s)t_(s)c_(s)a_(s)a_(s)t_(s)c_(s)c_(s)a_(s)t 55(c) 137 GGGCAACGTCGGGGCA 688

a_(s)a_(s)c_(s)g_(s)t_(s)c_(s)g_(s)g_(s)

689

a_(s)a_(s)c_(s)g_(s)t_(s)c_(s)g_(s)g_(s)

690

a_(s)a_(s)c_(s)g_(s)t_(s)c_(s)g_(s)g_(s)

691g_(s)g_(s)g_(s)c_(s)a_(s)a_(s)c_(s)g_(s)t_(s)c_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a 66(c) 138 TGCCAGGCAGGGGGCA 692

a_(s)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)

693 694

a_(s)a_(s)c_(s)g_(s)t_(s)c_(s)g_(s)g_(s)

695t_(s)g_(s)c_(s)c_(s)a_(s)g_(s)g_(s)c_(s)a_(s)g_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a140(c) 139 CCGGGGTGCAGGCGCA 696

g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g_(s)g_(s)

697

g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g_(s)g_(s)

698

g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g_(s)g_(s)

699c_(s)c_(s)g_(s)g_(s)g_(s)g_(s)t_(s)g_(s)c_(s)a_(s)g_(s)g_(s)c_(s)g_(s)c_(s)a148(c) 140 CATCCGCTCCGGGGTG 700

c_(s)g_(s)c_(s)t_(s)c_(s)c_(s)g_(s)g_(s)

701

c_(s)g_(s)c_(s)t_(s)c_(s)c_(s)g_(s)g_(s)

702

c_(s)g_(s)c_(s)t_(s)c_(s)c_(s)g_(s)g_(s)

703C_(s)A_(s)T_(s)C_(s)C_(s)G_(s)C_(s)T_(s)C_(s)C_(s)G_(s)G_(s)G_(s)G_(s)T_(s)G177(c) 141 GTGGGGCAGTGGATGA 704

g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)

705

g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)

706

g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)

707g_(s)t_(s)g_(s)g_(s)g_(s)g_(s)c_(s)a_(s)g_(s)t_(s)g_(s)g_(s)a_(s)t_(s)g_(s)a260(c) 142 CCTCTATGGGGTCGTC 708

t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)t_(s)

709

t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)t_(s)

710

t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)t_(s)

711c_(s)c_(s)t_(s)c_(s)t_(s)a_(s)t_(s)g_(s)g_(s)g_(s)g_(s)t_(s)c_(s)g_(s)t_(s)c274(c) 143 ATGCTTTTTATGTTCC 712

t_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)

713

t_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)

714

t_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)

715a_(s)t_(s)g_(s)c_(s)t_(s)t_(s)t_(s)t_(s)t_(s)a_(s)t_(s)g_(s)t_(s)t_(s)c_(s)c384(c) 144 GTTTGCTTTGCAATTT 716

c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)a_(s)

717

c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)a_(s)

718

c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)a_(s)

719g_(s)t_(s)t_(s)t_(s)c_(s)c_(s)t_(s)t_(s)t_(s)g_(s)c_(s)a_(s)a_(s)t_(s)t_(s)tISIS 145 TGTGCTATTCTGTGAATT 720

c_(s)t_(s)a_(s)t_(s)t_(s)c_(s)t_(s)g_(s)t_(s)g_(s)

23722 (18-mer) 721

G_(s)c_(s)t_(s)a_(s)t_(s)t_(s)c_(s)t_(s)g_(s)t_(s)g_(s)

722t_(s)g_(s)t_(s)g_(s)c_(s)t_(s)a_(s)t_(s)t_(s)c_(s)t_(s)g_(s)t_(s)g_(s)a_(s)a_(s)t_(s)t723

c_(s)t_(s)a_(s)t_(s)t_(s)c_(s)t_(s)g_(s)t_(s)g_(s)

146 724

c_(s)t_(s) g _(s)t_(s)t_(s)c_(s)t_(s) a _(s)t_(s)g_(s)

* 725

G _(s)c_(s)t_(s) g _(s)t_(s)t_(s)c_(s)t_(s) a _(s)t_(s)g_(s)

* 726

c_(s)t_(s) g _(s)t_(s)t_(s)c_(s)t_(s) a _(s)t_(s)g_(s)

* *relates to compound Underlined indicates mismatch compared to abovecompound. Compound 145F and 146F contains the MOE chemistry in capitalletters italic which is the compound ISIS23722.

TABLE 3 IC₅₀ (nM) of LNA (β-D-oxy-LNA) containing oligomeric in two celllines of different origin Oliqomeric compounds were evaluated for theirpotential to knockdown Survivin mRNA in 15PC3 and MCF7 cells. Transcriptsteady state was monitored by Real-time PCR and normalised to the GAPDHtranscript steady state. Seq ID/NO MCF7 15PC3  147 28 5  148 <5  155 <5 156 5  163 8 3  164 <5  175 11 3 199 1 <1 200 <1 203 1 612 <5 620 <25623 <5 652 <5 653 <25 656 <25 664 <25 665 <5

As showed In table 1 and 2, SEQ ID NO 2, 3, 4, 5, 6, 8, 9, 10, 11, 12,13, 14, 15, 118, 119, 120, 121, 122, 123, 125, 126, 128, 129, 130 and131 demonstrated at least 30% inhibition of survivin expression at 25 nMin these experiments and are therefore preferred.

Compounds of particular interest are 147, 148, 155, 156, 163, 164, 199,200, 203, 616, 617, 624, 625, 652, 653, 660, 661, 664 and 665.

Example 11 Improved Inhibition In Vitro of Survivin Expression Using LNAAntisense Oligomeric Compounds Compared to Phosphorothioates and MOE

Comparison of mRNA inhibition by using phosphorothioates and MOE(Calbiochem) versus LNA containing antisense oligomeric compound in15PC3 cells was performed. A LNA version of ISIS23722 was compared tothe MOE containing compound, which is an 18-mer 4MOE/PS+10PS+4MOE/PS andcompared to an iso-sequential phosphorthioate. Transfection wasperformed of 15PC3 with oligonucleotides or media (mock) (see example5). Survivin mRNA was monitored with realtime PCR and normalised toGAPDH. Survivin mRNA presented relative to mock expression (see Table4).

TABLE 4 Percentage down regulation of mRNA 0.2 nM 1 nM 5 nM 25 nM 100 nMLNA version of ISIS23722 <20%  48%  79%  84%  76% (4LNA/PS + 10PS +4LNA/PS): 145A MOE compound ISIS23722 <20% <20% <20% <20%  46%(4MOE/PS + 10PS + 4MOE/PS): 145F Phosphorthioate version  22% <20% <20%<20% <20% of ISIS23722 (18PS) 145D LNA version of ISIS23722 — — — <20%<20% with 6 mismatches: 146C MOE compound ISIS23722 — — — <20% <20% with6 mismatches: 146F

In another experiment, the supernatants from each culture well were alsoincluded in the analysis in order to allow late apoptotic cells to beanalyzed. The 18-mer LNA, PS and MOE compounds above were compared toLNA 16-mers of the invention. 15PC3 cells were transfected with theindicated oligos at the given concentrations (see Example 5). Total RNAwas extracted at 24 hours. Cells in the media supernatant were includedin the analysis. Survivin mRNA was monitored with realtime PCR andnormalised to GAPDH. Survivin mRNA presented relative to mockexpression. (see Table 5)

TABLE 5 Down regulation of mRNA (percentage of mock expression)Description: Seq ID 100 nM 25 nM 5 nM LNA version of ISIS23722 91% 94%89% (4LNA/PS + 10PS + 4LNA/PS): 145A LNA version of ISIS23722 89% 88%79% (4LNA/PO + 10PS + 4LNA/PO): 145C MOE compound ISIS23722 68% 36%<20%   (4MOE/PS + 10PS + 4MOE/PS): 145F Phosphorthioate version of 35%<20%   <20%   ISIS23722 (18PS): 145D LNA compound 2A (16-mer) 99% 90%66% LNA compound 6A (16-mer) — 98% 90% LNA compound 15B (16-mer) 97% 97%99%

Example 12 Apoptosis Induction by LNA Antisense Oligomeric Compounds

Cells were seeded to a density of 12000 cells per well in white 96 wellplate (Nunc 136101) in DMEM the day prior to transfection. The next daycells were washed once in prewarmed OptiMEM followed by addition of 72μl OptiMEM containing 5 μg/ml Lipofectamine 2000 (In vitrogen). Cellswere incubated for 7 min before adding 18 μl oligonucleotides diluted inOptiMEM. The final oligonucleotide concentration ranged from 5 nM to 25nM. After 4 h of treatment, cells were washed in OptiMEM and 100 μl DMEMcontaining serum was added. Following oligo treatment cells were allowedto recover for the period indicated before they were removed from theCO2 incubated and equilibrated to room temperature for 15 min. Thehighly sensitive Caspase 3/7-Glo™ Reagent (Promega) was added directlyto the cells in 96well and plates were incubated for 20 min beforerecording luminescence (luciferase activity) in Luminoskan Ascentinstrument from Thermo Labsystems after further 1 min lag period. Theluciferase activity is measured as Relative Light Units per seconds(RLU/s). The data was processed in the Ascent software 2.4.2, and graphswere drawn in excel. (see FIG. 8)

Example 13 Improved Induction of Apoptosis In Vitro Using LNA AntisenseOligomeric Compounds Compared to Phosphorothioates and MOE

Measurement of apoptosis using BD™ cytometric bead array (CBA) (cat.557816). Cells were transfected using lipofectamine 2000 as described(see Example 5). 24 h following transfection, the cells from thesupernatant was spun down and the adherent cells were trypsionised andspun down. The cell pellet was resuspended/washed in PBS and counted tobring cell concentration to 2×10⁶ cells/ml lysis buffer containingprotease inhibitors. The procedure was proceeded as described bymanufacturer with the following modifications. When cells were lysed,they were lysed for 40 min and vortexed with a 10 min interval. 1×10⁵cells were incubated with Caspase 3, Bcl-2 and PARP beads, mixed brieflyand incubated for 1 h at room temperature. Caspase 3 activity, Bcl2expression and induction of PARP in oligo treated cells were analysedusing the using the BD™ CBA software. Data were transferred to excel andgraphs were drawn. All data were-related to mock (which is set to one).FIG. 9 shows that the LNA containing compounds (145A and 145C) improvesinduction of apoptosis compared to the iso-sequential MOE compoundISIS27322 (here 145F) and the iso-sequential phosphorthioate compound(145D). Mismatch controls of a LNA compound (146C) and the MOE compound(146F) as well as the LNA compound 15A was also included in the study.Furthermore, Caspase 3 activation of compound 15A was detected byimmunohistochemical analysis of LNA oligomeric compound treated cells(FIG. 10).

Example 14 Antisense Oligonucleotide Inhibition of Survivin inProliferating Cancer Cells

Cells were seeded to a density of 12000 cells per well in white 96 wellplate (Nunc 136101) in DMEM the day prior to transfection. The next daycells were washed once in prewarmed OptiMEM followed by addition of 72μl OptiMEM containing 5 μg/ml Lipofectamine2000 (In vitrogen). Cellswere incubated for 7 min before adding 18 μl oligonucleotides diluted inOptiMEM. The final oligonucleotide concentration ranged from 5 nM to 100nM. After 4 h of treatment, cells were washed in OptiMEM and 100 μlserum containing DMEM was added. Following oligo treatment cells wereallowed to recover for the period indicated, viable cells were measuredby adding 20 μl the tetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES) (CellTiter 96® AQ_(ueous) One Solution CellProliferation Assay, Promega). Viable cells were measured at 490 nm in aPowerwave (Biotek Instruments). Growth rate (ΔOD/h) were plotted againstoligo concentration. (see FIG. 11).

Example 15 Measurement of Ploidy (Cell Cycle) and DNA Degradation(Apoptosis) of Cells Following Treatment with Oligomeric CompoundsTargeting Survivin

The late stage in the apoptotic cascade leads to large numbers of smallfragments of DNA which can be analysed by propidium iodide staining ofthe cells, furthermore, propidium iodide staining can be used to assesploidy in treated cells. To assess ploidy/apoptosis of cells treatedwith oligomeric compound directed against Survivin, cells were washed inPBA and fixed for 1 h in 70% EtOH at 4° C. After treatment with 50 μg/mlRNAse (Sigma) for 20 min at room temperature cells were washed with PBSand incubated with 40 μg/ml propidium iodide (Sigma or BD) for 30 min.All samples were analysed using fluorescence activated cell sorter(FACSCalibur, Becton Dickinson) and Cell Quest software. In the DNAhistogram the hypodiploid or the sub-G1 peak represented the apoptoticcells.

Example 16 Measurement of Changes in the Mitochondrial MembranePotential of Cells Following Treatment with Oligomeric CompoundsTargeting Survivin

To measure changes in the mitochondrial membrane potential theMitoSensor™ reagent method (Becton Dickinson, Cat # K2017-1) was used.MitoSensor™ reagent is taken up by healthy cells, in which it formsaggregates that emit red fluorescence. Upon apoptosis the mitochondrialmembrane potential changes and does not allow the reagent to aggregatewithin the mitochondria and therefore it remains in the cytoplasm in itsmonomeric form where it emits green fluorescence. Cells treated witholigomeric compounds directed against Survivin were washed and incubatedin MitoSensor Reagent diluted in Incubation buffer as described bymanufacturer. Changes in membrane potential following oligo treatmentwas detected by fluorescence activated cell sorter (FACSCalibur, BectonDickinson) and by the use of Cell Quest software.

Example 17 Inhibition of Capillary Formation of Endothelial CellsFollowing Antisense Oligo Treatment

Endothelial monolayer cells (e.g. HUVEC) were incubated with antisenseoligos directed against survivin. Tube formation was analysed by eitherof the two following methods. The first method was the BD BioCoatangiogenesis tube formation system. Cells were transfected with oligosas described (example 5). Transfected cells were seeded at 2×10⁴cells/96 well onto matrigel polymerized BD Biocoat angiogeneis plates.The plates were incubated for the hours/days indicated with or withoutPMA (5-50 nM), VEGF (20-200 ng/ml), Suramin or vehicle. The plates werestained with Cacein AM as stated by the manufacturer and images weretaken. Total tube length was measured using MetaMorph. Althernatively,cells were seeded in rat tail type I collagen (3 mg/ml, BectonDickinson) in 0.1 volume of 10×DMEM, neutralised with sterile 1 M NaOHand kept on ice or in matrigel. Cells were added to the collagensuspension at a final concentration of 1×10⁶ cells/ml collagen. Thecell-collagen mixture was added to 6-well or 35 mm plates and placed ina humidified incubator at 37° C. When gelled 3 ml of culture medium plusan extra 10% FBS were added and cells were allow to form capillary-likevascular tubes over the period indicated in the presence or absence ofPMA (16 nM), VEGF (50 ng/ml). Tube formation was quantified followingcryostat sectioning of the gels and examination of sections byphase-contrast microscopy.

Example 18 Measurement of In Vitro Cytotoxicity Following Treatment withOligomeric Compounds Targeting Survivin

Cells were seeded (0.3-1.2×10⁴) and treated with antisense oligos asdescribed (example for MTS assay Example 12). At the times indicated,20-50 μl medium from the antisense treated cells were transferred to96-well plates in order to measure the release of LDH to the medium. Anequal volume of LHD substrate was added as described by themanufacturer. Released LDH was measured using a 30-minute coupledenzymatic assay, which results in the conversion of a tetrazolium salt(INT) into a red formazan product. The amount of colour formed isproportional to the number of lysed cells. Visible wavelength absorbancedata (measured at 490 nm) were collected using a standard 96-well platereader (Powerwave, Bio-Tek Instruments). As positive control cells weretreated for about 45 minutes with 0.9% Triton X-100 (=100% lysis).Cytotoxicity was plotted relative to mock and Triton-x 100 treated cells(100% lysis=100% cytotoxicity).

Example 19 In Vivo Model: Tumour Growth Inhibition of Human Tumour CellsGrown In Vivo by Systemic Treatment with Antisense Oligonucleotides

Female NMRI athymic nude mice of 6 weeks old were purchased from M&B,Denmark and allowed to acclimatize for at least one week before enteringexperiments. Human cancer cells typically 10⁶ cells suspended in 300 μlmatrigel (BD Bioscience), were subcutaneously injected into the flanksof 7-8 week old NMRI athymic female nude mice. When the tumour growthwas established, typically 7-12 days post tumour cell injection;different antisense oligonucleotides were administrated at 5 mg/kg/dayfor up to 28 days using ALZET osmotic pumps implanted subcutaneously.Prior to dorsal implantation the pumps were incubated overnight at roomtemperature in sterile PBS to start the pumps. Control animals receivedsaline alone for the same period. Each experimental group included atleast 5 mice. Anti-tumour activities were estimated by the inhibition oftumour volume. Tumour growth was followed regularly by measuring 2perpendicular diameters. Tumour volumes were calculated according to theformula (π×L×D²/6), where L represents the largest diameter and D thetumour diameter perpendicular to L. At the end of treatment the animalswere sacrificed and tumour weights were measured. Mean tumour volume andweights of groups were compared using Mann-Whitney's test. All analysiswas made in SPSS version 11.0 for windows. Optimally a Western blotanalysis may also be performed to measure if the antisenseoligonucleotides have an inhibitory effect on protein levels. At the endof treatment period mice were therefore anaesthetised and the tumourswere excised and immediately frozen in liquid nitrogen. The tumours werehomogenized in lysis buffer (i.e. 20 mM Tris-Cl [pH 7.5]; 2% TritonX-100; 1/100 vol. Protease Inhibitor Cocktail Set III (Calbiochem);1/100 vol. Protease Inhibitor Cocktail Set II (Calbiochem)) at 4° C.with the use of a motor-driven homogeniser. 500 μl lysis buffer wasapplied per 100 mg tumour tissue. Tumour lysates from each group of micewere pooled and centrifuged at 13.000 g for 5 min at 4° C. to removetissue debris. Protein concentrations of the tumour extracts weredetermined using the BCA Protein Assay Reagent Kit (Pierce, Rockford).The protein extracts (50-100 μg) were fractionated on a gradientSDS-PAGE gel spanning from 4-20% and transferred to PVDF membranes andvisualized by aminoblack staining. The expression of survivin wasdetected with anti-human survivin antibody followed by horseradishperoxidase-conjugated anti-goat IgG (DAKO). Immunoreactivity wasdetected by the ECL Plus (Amersham biotech) and quantitated by aVersadoc 5000 lite system (Bio-Rad).

Example 20 In Vivo Model: Tumor Growth Inhibition of Human TumourFragments Transplanted in Nude Mice After Intraperetoneal Treatment withLNA Antisense Oligos

Tumour growth inhibiting activity of LNA antisense oligonucleotides wastested in xenotransplanted athymic nude mice, NMRI nu/nu, fromOncotest's (Freiburg, Germany) breeding colony. Human tumour fragmentsfrom breast (MDA MB 231), prostate (PC3) or lung tumours (LXFE 397,Oncotest) were obtained from xenografts in serial passage in nude mice.After removal of tumors from donor mice, they were cut into fragments(1-2 mm diameter) and placed in RPMI 1640 culture medium untilsubcutaneous implantation. Recipient mice were anaesthetized byinhalation of isoflurane. A small incision was made in the skin of theback. The tumor fragments (2 fragments per mouse) were transplanted withtweezers. MDA MB 231 and LXFE 397 tumors were transplanted in femalemice, PC3 tumors were transplanted in male mice. When a mean tumourdiameter 4-6 mm was reached, animals were randomized and treated witholigonucleotides at 20 mg/kg intraperetoneally once a day for threeweeks excluding weekends. A vehicle (saline) and positive control group(Taxol, 20 mg/kg/day) were included in all experiments. All groupsconsisted of 6 mice. The tumour volume was determined by two-dimensionalmeasurement with a caliper on the day of randomization (Day 0) and thentwice weekly. Tumor volumes were calculated according to the formula:(a×b²)×0.5 where a represents the largest and b the perpendicular tumordiameter. Mice were observed daily for 28 days after randomization untiltumour volume was doubled. Mice were sacrificed when the tumourdiameters exceeded 1.6 cm. For the evaluation of the statisticalsignificance of tumour inhibition, the U-test by Mann-Whitney-Wilcoxonwas performed. By convention, p-values <0.05 indicate significance oftumor inhibition.

Example 21 Biodistribution of Oligonucleotides in Mice

Female NMRI athymic nude mice of 6 weeks old were purchased from M&B,Denmark and allowed to acclimatize for at least one week before enteringexperiments. Human cancer cells typically 10⁶ cells suspended in 300 μlmatrigel (BD Bioscience) were subcutaneously injected into the flanks of7-8 week old NMRI athymic female nude mice. When tumour growth wasevident, tritium labelled oligonucleotides were administrated at 5mg/kg/day for 14 days using ALZET osmotic pumps implantedsubcutaneously. The oligonucleotides were tritium labeled as describedby Graham M J et al. (J Pharmacol Exp Ther 1998; 286(1): 447-458).Oligonucleotides were quantitated by scintillation counting of tissueextracts from all major organs (liver, kidney, spleen, heart, stomach,lungs, small intestine, large intestine, lymph nodes, skin, muscle, fat,bone, bone marrow) and subcutaneous transplanted human tumour tissue.

Example 22 Uptake of LNA Oligomeric Compound in Human Tumour Xenografts

Human 15PC3 xenografted tumors according to Example 13 were homogenizedin 10 volumes of 0.5% Igepal CA-630, 25 mM Tris pH 8.0, 25 mM EDTA, 100mM NaCl, 1 mg/m1 Proteinase K1 and incubated overnight at 37 degreescelsius followed by phenol-chloroform extraction. The concentration ofantisense oligonucleotide 2650 in the combined aqueous phase wasdetermined using a sequence specific ELISA assay. Two probes, onelabelled with biotin and one labelled with digoxigenin (DIG) withcomplementary sequences to the antisense oligonucleotide are hybridisedto the antisense oligo. The complex is captured by immobilizedstreptavidin and quantified using a horse radish peroxidase-conjugatedanti-digoxigenin antibody and standard ELISA procedures. Briefly, 10 nMDNA capture probe (5′-aactgtgc-Biotin-3′) and 10 nM LNA detection probe(5′-DIG-GATGTTTCgatgtttc-3′)(SEQ ID NO: 738) were mixed with sample orstandards in 1% blocking reagent (Roche cat. 1 096 176) in PBS. Theprobes were annealed to the oligo by heating the mixture to 70 degreescelsius and gradual cooling to 20 degrees Celsius. The mixture wastransferred to streptavidin-coated wells. The amount of capturedDIG-probe is quantified using an HRP-conjugated Anti-DIG antibodyfragment (Roche) and standard ELISA procedures. At least 1.3 μg/gtumours tissue of the oligomeric compound 15A was detected (data notadjusted for recovery).

The present invention has been described with specificity in accordancewith certain of its preferred embodiments. Therefore, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

1. A method for treating prostatic, breast or lung carcinoma, saidmethod comprising administering an effective amount of anoligonucleotide compound having the sequence as defined in SEQ ID NO:130 to a patient in need thereof.
 2. The method according to claim 1,wherein said carcinoma is.
 3. The method according to claim 1, whereinsaid carcinoma is breast carcinoma.
 4. The method according to claim 1,wherein said carcinoma is lung carcinoma.
 5. The method according toclaim 1, wherein the oligonucleotide compound has the sequence asdefined in SEQ ID NO:
 660. 6. The method according to claim 1, whereinthe oligonucleotide compound has the sequence as defined in SEQ ID NO:661.
 7. The method according to claim 1, wherein the oligonucleotidecompound has the sequence as defined in SEQ ID NO:
 662. 8. The methodaccording to claim 1, wherein the oligonucleotide compound has thesequence as defined in SEQ ID NO:
 663. 9. The method according to claim1, wherein the oligonucleotide compound is administered parenterally.10. The method according to claim 1, wherein the oligonucleotidecompound is administered intravenously.
 11. The method according toclaim 1, wherein the oligonucleotide compound is administered by bolusinjection into a target organ.
 12. The method according to claim 1,wherein the oligonucleotide compound is administered intraperitoneally.